Method of stimulating an immune response using a pseudotyped lentivirus

ABSTRACT

Methods and compositions are provided for delivery of a polynucleotide encoding a gene of interest, typically an antigen, to a dendritic cell (DC). The virus envelope comprises a DC-SIGN specific targeting molecule. The methods and related compositions can be used to treat patients suffering from a wide range of conditions, including infection, such as HIV/AIDS, and various types of cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.13/887,908, filed May 6, 2013, now U.S. Pat. No. 8,906,359, which is acontinuation of U.S. application Ser. No. 12/688,689, filed Jan. 15,2010, now U.S. Pat. No. 8,715,640, which is a continuation of U.S.application Ser. No. 11/781,865, filed Jul. 23, 2007, now U.S. Pat. No.8,329,162, which claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/832,497, filed Jul. 21, 2006 and U.S.Provisional Application No. 60/920,260, filed Mar. 27, 2007, each ofwhich is herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

The invention was made with government support under Grant No. AI068978awarded by the National Institutes of Health. The government has certainrights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“SeqList_CALTE-035C1.txt”, created Jan. 13, 2010, which is 132,038 bytesin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The invention relates generally to targeted gene delivery, and moreparticularly to the use of a recombinant virus comprising a targetingmolecule that targets and binds dendritic cells and can thus be used fordendritic cell vaccination.

Immunization is one of the most productive tools in modern medicalpractice but remains burdened by limitations. Certain infectiousdiseases such as HIV/AIDS, malaria, and tuberculosis are not currentlycontrolled at all by immunization, while other infectious diseases arecontrolled by complex immunization regimens. Cancer is a promisingtarget for immunotherapeutic treatments, but clinical outcomes inexperimental trials have been disappointing (Rosenberg, S. A. et al.2004. Nat. Med. 10:909-915, which is incorporated herein by reference inits entirety). Novel methods of immunization are needed, for example, toreliably induce anti-tumor immunity.

Dendritic cells (DCs) play critical roles in both innate and adaptiveimmunity. DCs are specialized antigen-presenting cells with the uniquecapability to capture and process antigens, migrate from the peripheryto a lymphoid organ, and present the antigens to resting T cells in amajor histocompatibility complex (MHC)-restricted fashion (Banchereau,J. & Steinman, R. M. 1998. Nature 392:245-252; Steinman, R. M., et al.2003. Ann Rev Immunol 21: 685-711, each of which is incorporated hereinby reference in its entirety). These cells are derived from bone marrow(BM) and are characterized by dendritic morphology and high mobility.Immature DCs are adept at antigen ingestion and are distributed assentinels in peripheral tissue throughout the body. However, maturationof DCs is required in order to mount an efficient immune response(Steinman, R. M., et al. 2003. supra). The matured DCs express highlevels of MHC-antigen complex and other costimulatory molecules (such asCD40, B7-1, B7-2 and CD1a) (Steinman, R. M. 1991. Ann Rev Immunol 9:271-296, which is incorporated herein by reference in its entirety;Banchereau, J. and R. M. Steinman. 1998. supra). These molecules playkey roles in stimulating T cells.

The discovery of the role of DCs as specialized antigen-presenting cells(APCs) has fueled attempts at DC-based immunization/vaccination thatinvolve loading DCs with specific antigens (Banchereau, J. & Palucka, A.K. 2005. Nat. Rev. Immunol. 5:296-306; Figdor, C. G. et al. 2004. Nat.Med. 10:475-480, each of which is incorporated herein by reference inits entirety). However, all of these attempts involve ex vivo loading ofDCs with specific antigens. Ex vivo generated DCs are then administeredto the patient. Ex vivo generation of DCs for each patient is extremelylabor intensive process.

By contrast, the present invention is directed inter alia to targeting,antigen loading and activation of DCs in vivo, which results in vivotreatment of diseases by generating a beneficial immune response in thepatient. The invention thus fulfills a longstanding need for effectiveand efficient regimes for immunization/vaccination.

SUMMARY OF THE INVENTION

In one aspect of the invention methods of delivering a polynucleotide toa dendritic cell expressing DC-SIGN are provided. In some embodimentsthe methods comprise transducing the dendritic cell with a recombinantvirus, wherein the recombinant virus comprises the polynucleotide to bedelivered and a targeting molecule that binds DC-SIGN. In someembodiments the targeting molecule is specific for DC-SIGN.

In some embodiments of the invention, the recombinant virus comprisessequences from a lentivirus genome, such as an HIV genome.

In other embodiments the recombinant virus comprises sequences from agammaretrovirus genome, such as sequences from a Mouse Stem Cell Virus(MSCV) genome or a Murine Leukemia Virus (MLV) genome.

In some embodiments of the invention, the methods utilize a targetingmolecule comprising a viral glycoprotein derived from at least one virusselected from the group of: Sindbis virus, influenza virus, Lassa fevervirus, tick-borne encephalitis virus, Dengue virus, Hepatitis B virus,Rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Bornadisease virus, Hantaan virus, and SARS-CoV virus. In more particularembodiments, the targeting molecule comprises a modified viralglycoprotein derived from Sindbis virus (SIN or SVG). In certainembodiments, the targeting molecule is SINmu also known as SVGmu (SEQ IDNO: 11).

In some embodiments, the polynucleotide to be delivered to a dendriticcell comprises at least one of the following: a gene encoding a proteinof interest, a gene encoding a siRNA, and a gene encoding a microRNA.The gene encoding a protein of interest may encode an antigen, such as atumor antigen or an HIV antigen.

The recombinant virus may be produced by transfecting a packaging cellline with a viral vector comprising the polynucleotide to be deliveredand a vector comprising a gene encoding the targeting molecule;culturing the transfected packaging cell line; and recovering therecombinant virus from the packaging cell culture. In some embodiments,the packaging cell line is a 293 cell line.

In some embodiments of the invention, the polynucleotide is delivered toa dendritic cell in vitro, while in other embodiments the polynucleotideis delivered to a dendritic cell in a subject in vivo. The subject istypically a mammal, such as a human, mouse or guinea pig.

In another aspect, recombinant virus comprising: a polynucleotide ofinterest; and a targeting molecule that binds DC-SIGN are provided. Insome embodiments the targeting molecule specifically binds DC-SIGN. Therecombinant virus may comprise sequences from a lentivirus genome, suchas sequences from an HIV genome. In other embodiments the recombinantvirus comprises sequences from a gammaretrovirus genome, such assequences from a Mouse Stem Cell Virus (MSCV) genome or a MurineLeukemia Virus (MLV) genome.

The targeting molecule may comprise a viral glycoprotein derived from atleast one virus selected from the group of: Sindbis virus, influenzavirus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus,Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus,Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus. Insome embodiments the targeting molecule is a viral glycoprotein derivedfrom Sindbis virus. In particular embodiments, the targeting molecule isSVGmu (SEQ ID NO: 11).

The polynucleotide may be, for example, at least one of the following: agene encoding a protein of interest, a gene encoding a siRNA, and a geneencoding a microRNA of interest. In some embodiments the polynucleotideencodes an antigen, such as a tumor antigen or an HIV antigen.

In another aspect, methods of stimulating an immune response in a mammalare provided. A polynucleotide encoding an antigen to which an immuneresponse is desired is delivered to dendritic cells expressing DC-SIGNby contacting the dendritic cells with a recombinant virus comprisingthe polynucleotide and a targeting molecule that binds DC-SIGN. In someembodiments the targeting molecule is specific for DC-SIGN and does notbind appreciably to other molecules. In other embodiments the targetingmolecule binds preferentially to DC-SIGN.

In a further aspect, vectors encoding targeting molecules that bindDC-SIGN are provided. In some embodiments, the targeting molecule is amodified viral glycoprotein. In further embodiments, the targetingmolecule is SVGmu (SEQ ID NO: 11). The targeting molecule specificallybinds DC-SIGN in some embodiments. The vector may additionally encodeone or more genes of interest, such as a gene encoding an antigen and/ora gene encoding a dendritic cell maturation factor.

In a still further aspect, methods of treating a patient with a diseaseare provided. A recombinant virus is administered to the patient, wherethe recombinant virus comprises a polynucleotide encoding an antigenassociated with the disease and a targeting molecule that binds DC-SIGN.The targeting molecule may be derived from a viral glycoprotein. In someembodiments, the targeting molecule is SVGmu (SEQ ID NO: 11).

The disease to be treated is generally one for which an antigen is knownor can be identified. In some embodiments of the invention, the diseaseto be treated is cancer. In other embodiments, the disease is HIV/AIDS.

Dendritic cells transduced with a recombinant virus are also provided,where the recombinant virus comprises a polynucleotide of interest and atargeting molecule that binds DC-SIGN. In some embodiments, thetargeting molecule comprising a viral glycoprotein derived from at leastone virus selected from the group of: Sindbis virus, influenza virus,Lassa fever virus, tick-borne encephalitis virus, Dengue virus,Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus,Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus. Insome embodiments, the targeting molecule is SVGmu (SEQ ID NO: 11).

Further, methods of immunizing a mammal by delivering a polynucleotideencoding an antigen to dendritic cells expressing DC-SIGN are alsoprovided in which the dendritic cells are contacted with a recombinantvirus comprising a polynucleotide encoding an antigen and a targetingmolecule that binds DC-SIGN. In some embodiments, the dendritic cellsare contacted with the recombinant virus ex vivo. In other embodiments,the dendritic cells are contacted with the recombinant virus in vivo.

The methods disclosed herein can also be used to stimulate an immuneresponse to a specific antigen in a mammal by delivery of apolynucleotide encoding the antigen to dendritic cells using arecombinant virus comprising the polynucleotide and a targeting moleculethat binds DC-SIGN. The immune response may be modulated by providing afurther polynucleotide whose expression in the dendritic cell modulatesthe immune response. For example, a polynucleotide encoding a dendriticmaturation factor may be delivered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a general strategy to targetdendritic cells (DCs) for antigen delivery. Sindbis virus wild-typeglycoprotein is mutated at the heparan sulfate binding site to abolishits binding ability. The resulting mutant glycoprotein (SVGmu) bindsDC-SIGN but does not bind heparin sulfate. DC-SIGN: Dendritic CellSpecific ICAM-3 (Intracellular Adhesion Molecules 3)-GrabbingNonintegrin.

FIG. 2 illustrates laser confocal microscope images of virus particlesharvested from virus-producing cells transiently transfected withlentiviral vector, plasmids encoding GFP-vpr and SVGmu, and othernecessary packaging constructs. The virus particles are labeled with GFP(green). The surface incorporation of SVGmu was detected byimmunostaining with an anti-HA tag antibody (red) to label SVGmu. In the“GFP” slide, viral particles tabled with GFP are green. In the “SVGmu”slide, viral particles with surface incorporation of SVGmu are stainedred. In the “Merged” slide, viral particles where only GFP is expressedare green, viral particles where only SVGmu is incorporated into thesurface are red, and viral particles expressing both GFP and containingSVGmu are yellow. The overlay of the green and red colors (yellow)indicates the viral particles containing SVGmu, which represent themajority of the total virus particles. The scale bar represents 2 pm.

FIG. 3A shows flow cytometric analysis of constructed target cell lines293T.hDCSIGN expressing human DC-SIGN, and 293T.mDCSIGN expressingmurine DC-SIGN. Solid line: expression of DC-SIGN in target cell lines;shaded area: background staining in 293T cells.

FIG. 3B shows flow cytometry results for detection of GFP expressed in293T cells transduced with lentivector enveloped with wild-type Sindbisglycoprotein (FUGW/SVG) or mutant Sindbis glycoprotein (FUGW/SVGmu). Onemilliliter of fresh viral supernatants of FUGW/SVG and FUGW/SVGmu wereused to transduce 293T cells (2×10⁵) expressing human DC-SIGN(293T.hDCSIGN) or murine DC-SIGN (293T.mDCSIGN). The parental 293T cellslacking the expression of DC-SIGN were included as controls. Asillustrated, lentivector enveloped with the mutant Sindbis virusglycoprotein (SVGmu) is able to specifically transduce 293T cellsexpressing human or mouse DC-SIGN. The specific transduction titer ofFUGW/SVGmu was estimated to be approximately 1×10⁶ TU/ml for293T.hDC-SIGN and approximately 0.8×10⁶ TU/ml for 293T.mDC-SIGN.

FIG. 4A shows flow cytometry results that illustrate the ability of theFUGW lentivirus enveloped with the mutant Sindbis glycoprotein(FUGW/SVGmu) to specifically transduce mouse dendritic cells expressingDC-SIGN in a primary mixed bone marrow culture. Whole bone marrow cellsisolated from B6 mice were exposed to the fresh viral supernatant ofFUGW/SVGmu. The FUGW lentivector pseudotyped with the ecotropicglycoprotein (FUGW/Eco) was included as a non-targeting control. Surfaceantigens of the GFP-positive cells were assessed by staining withanti-CD11c and anti-DC-SIGN antibodies.

FIG. 4B shows flow cytometry results indicating that FUGW lentivirusenveloped with the mutant Sindbis glycoprotein (FUGW/SVGmu) does nottransduce other cell types including primary T cells (CD3⁺, top panel)and B cells (CD19^(±), bottom panel). Primary CD3⁺ T cells and CD19⁺ Bcells were isolated from the mouse spleen and transduced with the freshviral supernatant of either the targeting FUGW/SVGmu or non-targetingFUGW/Eco vector. GFP expression was analyzed by flow cytometry. Solidline: cells exposed to indicated lentiviral vector; shaded area: cellswithout transduction (as a negative control).

FIG. 5 shows flow cytometry results that illustrate the ability of theFUGW lentivirus enveloped with the mutant Sindbis glycoprotein(FUGW/SVGmu) to specifically transduce bone marrow-derived DCs (BMDCs).BMDCs were generated by culturing freshly isolated bone marrow cells inthe presence of cytokine GM-CSF for 6 days. The cells were thentransduced with the fresh viral supernatant of either the targetingFUGW/SVGmu or non-targeting FUGW/Eco vector. GFP and CD11c expressionwere measured by flow cytometry.

FIG. 6 shows activation of BMDCs after targeted transduction withFUGW/SVGmu. DC activation was assessed by analyzing the surfaceexpression of CD86 and I-A^(b) using flow cytometry. The addition of LPS(1 Kg/ml) overnight was used as a synergistic stimulator for theactivation of transduced BMDCs. Shaded area: GFP negative(untransduced); solid line: GFP positive (transduced).

FIG. 7A, FIG. 7B and FIG. 7C illustrate targeting of DCs in vivo usingFUGW/SVGmu lentivirus. B6 mice were injected with 50×10⁶ TU ofFUGW/SVGmu and analyzed 3 days later. Non-immunized mice were includedas a control. In FIG. 7A, the images show the size of a representativeinguinal lymph node close to the injection site compared to that of theequivalent lymph node distant from the injection site. FIG. 7Billustrates the total cell number counts of the indicated lymph nodes inFIG. 7A. FIG. 7C illustrates representative flow cytometric analysis ofCD11c⁺ cells from the two lymph nodes shown in FIG. 7A. The numbersindicate the fraction of GFP⁺ DC populations

FIG. 8 provides a schematic representation of the lentivector encodingthe OVA antigen (FOVA) (top) and the lentivector encoding GFP (FUGW) asa control (bottom).

FIG. 9 illustrates in vitro stimulation of CD8⁺ OT1 T cells by dendriticcells that were transduced with the FOVA/SVGmu (DC/FOVA) or FUGW/SVGmulentivector (DC/FUGW), or by non-transduced BMDCs pulsed with OVAppeptide (SIINFEKL) (DC/OVAp). Patterns of surface activation markers ofOT1 T cells cocultured with BMDCs were assessed by antibody staining forCD25, CD69, CD62L, and CD44. Shaded area: naïve OT1 T cells harvestedfrom transgenic animals; solid line: OT1 T cells cocultured withindicated BMDCs.

FIG. 10A illustrates the measurement of IFN-y by ELISA in OT1 T cellsmixed with various dilutions of BMDCs transduced with FOVA/SVGmu (.),FUGW/SVGmu (P), or pulsed with OVAp peptide (•) and cultured for 3 days.

FIG. 10B illustrates the proliferative responses of treated OT1 T cellsfrom FIG. 10A measured by a [³H] thymidine incorporation assay for 12hours.

FIG. 11 illustrates in vitro stimulation of CD4⁺ OT2 T cells bydendritic cells that were transduced with the FOVA/SVGmu (DC/FOVA) orFUGW/SVGmu lentivector (DC/FUGW), or by non-transduced BMDCs pulsed withOVAp* peptide (ISQAVHAAHAEINEAGR) (DC/OVAp*). Patterns of surfaceactivation markers of OT2 transgenic T cells cocultured with BMDCs wereassessed by antibody staining for CD25, CD69, CD62L, and CD44. Shadedarea: naïve OT2 T cells harvested from transgenic animals; solid line:OT2 T cells cocultured with BMDCs.

FIG. 12 illustrates the measurement of IFN-y by ELISA in OT2 T cellsmixed with various dilutions of BMDCs transduced with FOVA/SVGmu (a),FUGW/SVGmu (.), or pulsed with OVAp* peptide (•) and cultured for 3days.

FIG. 13A provides a schematic representation of the retroviral vectorMIG-0T1 used for genetic modification of murine hematopoietic stem cells(HSCs).

FIG. 13B illustrates how CD8⁺ OT1 T cells derived from theMIG-0T1-modified HSCs in reconstituted mice were identified by theco-expression of GFP and TCR Va2 or V135. HSCs from B6 mice wereinfected with MIG-0T1 pseudotyped with Eco (MIG-OT1/Eco) and transferredinto irradiated B6 recipient mice. Eight weeks post-transfer, the CD8⁺OT1 T cells were identified by flow cytometry.

FIG. 14A illustrates assessment of patterns of surface activationmarkers on GFP^(±)OT1⁺ T cells isolated from the spleens ofreconstituted and immunized mice. Mice reconstituted by MIG-0T1 modifiedHSCs were immunized by direct subcutaneous injection of 10×10⁶ TU ofeither FOVA/SVGmu or FUGW/SVGmu (as a control) and analyzed seven dayslater. Detection of surface staining for CD69, CD62L and CD44 wasconducted. Solid line: GFP^(±)OT1⁺ T cells from FOVA/SVGmu-immunizedmice; dotted line: GFP⁺OT1⁺ T cells from control FUGW/SVGmu-immunizedmice; shaded area: GFP^(±)OT1⁺ T cells from non-immunized mice.

FIG. 14B illustrates the total number of OT 1 cells harvested from lymphnodes (LN, □) or spleens (SP, ▪) of non-immunized mice (no imm) or miceimmunized with FUGW/SVGmu or FOVA/SVGmu.

FIG. 15 illustrates in vivo stimulation of antigen specific T cell andantibody responses in wild-type mice following a subcutaneous injectionof the DC-targeting lentivector FOVA/SVGmu. B6 mice were immunizedsubcutaneously with 50×10⁶ TU of either FOVA/SVGmu or FUGW/SVGmu (as acontrol). Mice without immunization (no imm.) were included as anegative control. Fourteen days post-immunization, spleen cells wereharvested and analyzed for the presence of OVA-specific T cells measuredby H-2K^(b)-SIINFEKL-PE tetramer and CD44 staining. Indicatedpercentages are the percent of total CD8⁺ T cells.

FIG. 16A and FIG. 16B illustrate in vivo OVA-specific T cell responsesseen in mice receiving different subcutaneous doses of FOVA/SVGmu.OVA-specific T cells were identified by tetramer staining as in FIG. 17.FIG. 16A shows the measured percentage of OVA-specific T cells followingimmunization with 100×10⁶ TU of FOVA/SVGmu. FIG. 16B shows the doseresponses of OVA-specific T cells following injection of the indicateddoses of FOVA/SVGmu.

FIG. 17A illustrates the patterns of surface activation markers ofOVA-specific CD8⁺ T cells (identified as tetramer positive cells)isolated from FOVA/SVGmu immunized mice 2 weeks post-injection. Thesurface activation markers were assessed by antibody staining for CD25,CD69, CD62L and CD44. Solid line: tetramer⁺CD8⁺ T cells fromFOVA/SVGmu-immunized mice; shaded area: naïve CD8⁺ T cells fromnon-immunized mice.

FIG. 17B illustrates the OVA-specific serum IgG titer of B6 micefollowing immunization with 50×10⁶ TU FOVA/SVGmu. Sera were collected onday 7 and day 14 post-immunization and were analyzed for the titer ofOVA-specific IgG using ELISA at serial 10× dilutions, starting at 1:100.The titer values were determined by the highest dilution at which theoptical density was 2× standard derivations higher than that of thebaseline serum at the equivalent dilution.

FIG. 18 illustrates tumor size as a function of time in a murine E.G7tumor model. B6 mice were immunized with subcutaneous injection of50×10⁶ TU of either FOVA/SVGmu (A) or mock vector FUW/SVGmu (.). Noimmunization (▪) was included as a control. Four mice were included ineach group. At day 14 post-immunization, the mice were challenged with5×10⁶ of either E.G7 tumor cells (expressing the OVA antigen, leftpanel) or the parental EL4 tumor cells (lacking the OVA antigen, as acontrol, right panel) subcutaneously. Tumor growth was measured with afine caliper and is shown as the product of the two largestperpendicular diameters (mm²).

FIG. 19 illustrates the in vivo the kinetic growth of tumors in a murineE.G7 tumor eradication model. An albino strain of B6 mice were implantedwith 5×10⁶ E.G7 tumor cells stably expressing a firefly luciferaseimaging gene (E.G7.1uc). A mouse (#1) without tumor implantation wasincluded as a control. Mice bearing tumors were treated withoutimmunization (#2), or with immunization by the injection of 50×10⁶ TU ofFOVA/SVGmu at days 3 and 10 (#3, #4) post tumor challenge. The kineticgrowth of the tumors was monitored by live animal imaging using BLI. Thep/s/cm²/sr represents photons/s ec/cm²/steri di an.

FIG. 20 shows the quantitation of luminescence signals generated by theE.G7 tumors in FIG. 19. (□) for mouse #2; (to) for mouse #3; (A) formouse #4.

FIG. 21 illustrates the percentage of OVA-specific T cells presentfollowing immunization with 100×10⁶ TU of FOVA/SVGmu in the albinostrain of B6 mice. Albino B6 mice were immunized subcutaneously with50×10⁶ TU of FOVA/SVGmu. Mice without immunization (no imm.) wereincluded as a negative control. Fourteen days post-immunization, spleencells were harvested and analyzed for the presence of OVA-specific Tcells measured by H-2K^(b)-SIINFEKL-PE tetramer and CD44 staining.Indicated percentages are the percent of total CD8⁺ T cells.

FIG. 22A provides a schematic representation of a DC-targetedlentivector encoding an imaging gene firefly luciferase (Luc),designated as Fluc/SVGmu.

FIG. 22B illustrates bioluminescence imaging of mice injectedsubcutaneously with 50×10⁶ TU of either the DC-targeting Fluc/SVGmulentivector (shown in FIG. 25A) or a non-targeting Fluc/VSVGlentivector. The representative image was obtained at day 30post-injection using IVIS200® (Xenogen).

FIG. 23 illustrates that administration of a single dose of recombinantDC-specific lentivector FOVA/SVGmu can generate IFN-y⁺CD8⁺ T cells in B6mice. Naive B6 mice are immunized by subcutaneous injection of 50×10⁶ TUof FOVA/SVGmu lentivector, or the same dose of FUGW/SVGmu as a control.The non-immunized B6 mice (no imm.) were included as a negative control.Two weeks later, spleen cells were harvested from the experimental mice,and were analyzed for intracellular IFN-y production using flowcytometry with or without OVAp peptide restimulation. Indicatedpercentages are the percent of IFN-y⁺CD8⁺ T cells of the total CD8⁺ Tcells.

FIG. 24 illustrates a schematic representation of lentiviral constructsfor preparation of DC-targeting recombinant viruses.

FIG. 25 shows a schematic representation of an embodiment of in situvaccination against HIV/AIDS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Genetic engineering has been shown to be an efficient and potent meansto convert dendritic cells (DCs) into special immune cells to induceantigen-specific immune responses. A great deal of research involving invitro manipulation of DCs for vaccination/immunization against cancer,HIV and other diseases has been conducted. However, until now, it hasnot been possible to specifically and efficiently deliver a gene ofinterest, such as a gene encoding an antigen, to dendritic cells invitro and in vivo. The inventors have discovered novel methods andcompositions for efficient and specific targeting of DCs in vitro and invivo. The methods and compositions can be used to induceantigen-specific immune responses, for example for immunotherapy.

Embodiments of the invention include methods and compositions fortargeting dendritic cells (DCs) by using a recombinant virus to delivera polynucleotide to the DCs. This is preferably accomplished throughtargeting the DC-specific surface molecule DC-SIGN (Dendritic CellSpecific ICAM-3 (Intracellular Adhesion Molecules 3)-GrabbingNonintegrin; also known as CD209). DC-SIGN is a C-type lectin-likereceptor capable of rapid binding and endocytosis of materials(Geijtenbeek, T. B., et al. 2004. Annu. Rev. Immunol. 22: 33-54, whichis incorporated herein by reference in its entirety). In preferredembodiments, recombinant viruses are enveloped with a designed targetingmolecule that is specific in its recognition for DC-SIGN. Thepolynucleotide can include, but is not limited to, a gene of interest,siRNA(s), and/or microRNA(s). In preferred embodiments, thepolynucleotide encodes an antigen. In some embodiments, the recombinantvirus delivers more than one gene to DCs. For example, genes encodingtwo or more antigens could be delivered. The delivery of more than onegene can be achieved, for example, by linking the genes with an InternalRibosome Entry Site (IRES), and/or with 2A sequences, and driving theexpression using a single promoter/enhancer.

As discussed in more detail below, embodiments of the invention arebased on the use of recombinant viruses, such as lentiviruses andgammaretroviruses, because these viruses are able to incorporate intotheir envelope a large number of proteins are found on the surface ofvirus-producing cells. However, as also discussed below, other types ofviruses may be used and the methods modified accordingly. Generally, apackaging cell line is transfected with a viral vector encoding apolynucleotide of interest (typically encoding an antigen), at least oneplasmid encoding virus packaging components (such as gag and poll and atargeting molecule that is engineered to bind dendritic cells. Inpreferred embodiments, the targeting molecule is genetically engineeredto specifically bind the DC-SIGN cell surface marker of dendritic cells.During budding of the virus, the targeting molecule, which is expressedin the packaging cell membrane, is incorporated into the viral envelope.As a result, the retroviral particles comprise a core including thepolynucleotide of interest and an envelope comprising the targetingmolecule on its surface.

The targeting molecule is able to bind DC-SIGN on a dendritic cell, andthe virus is able to deliver the gene of interest to the dendritic cell.Without wishing to be bound by theory, it is believed that the bindinginduces endocytosis, bringing the virus into an endosome, triggeringmembrane fusion, and allowing the virus core to enter the cytosol.Following reverse transcription and migration of the product to thenucleus, the genome of the virus integrates into the target cell genome,incorporating the polynucleotide of interest into the genome of thetarget cell. The DC then expresses the polynucleotide of interest(typically encoding an antigen). The antigen is then processed andpresented to T and B cells by DCs, generating an antigen-specific immuneresponse. The specific pathway described above is not required so longas the dendritic cell is able to stimulate an antigen-specific immuneresponse.

Embodiments of the present invention include methods and compositionsfor direct targeting of a gene of interest to DCs both in vitro and invivo. In some preferred in vivo embodiments, the gene of interest isdelivered to DCs without in vitro culture of DCs. For example, the geneof interest may be delivered to DCs via a direct administration of thetargeting virus into a living subject. The gene of interest preferablyencodes an antigen against which an immune response is desired.Exemplary antigens include: tumor specific antigens, tumor-associatedantigens, tissue-specific antigens, bacterial antigens, viral antigens,yeast antigens, fungal antigens, protozoan antigens, parasite antigens,mitogens, and the like. Other antigens will be apparent to one of skillin the art and can be utilized without undue experimentation.

The methods disclosed herein may be readily adopted to utilize targetingmolecules that are specific for DCs or that can be manipulated toprovide the desired specificity. The targeting molecule is preferably anengineered viral glycoprotein that binds DC-SIGN in dendritic cells andthat facilitates delivery of the gene of interest into the dendriticcells. Exemplary targeting molecules include, but are not limited to,glycoproteins derived from the following: Sindbis virus, influenzavirus, Lassa fever virus, tick-borne encephalitis virus, Dengue virus,Hepatitis B virus, Rabies virus, Semliki Forest virus, Ross River virus,Aura virus, Borna disease virus, Hantaan virus, and SARS-CoV virus. Thetargeting molecule is preferably membrane bound. If necessary, aDC-SIGN-specific targeting molecule that is designed or derived from aviral glycoprotein for use in the recombinant virus can be modified to amembrane bound form.

Any method known in the art can be used to engineer the targetingmolecule to provide the desired specificity. Exemplary methods include,but are not limited to, rational protein engineering and DNA shuffling.Generally, to engineer a targeting molecule specific for DCs, a viralglycoprotein that interacts with a dendritic cell-specific surfacemarker is provided. Preferably, the viral glycoprotein interacts withDC-SIGN. The viral glycoprotein can also interact with at least a secondcell surface marker such as, for example, heparin sulfate (HS), which isexpressed on cell types other than DCs. The viral glycoprotein ismodified such that its ability to interact with the DC-specific surfacemarker is maintained while its ability to interact with additional cellsurface markers is decreased or eliminated. The modification can be amutation in at least one residue of the viral glycoprotein amino acidsequence. The mutation can be a deletion, addition or substitution ofthe residue, and it can be carried out by standard methods known in theart. The desired specificity can readily be confirmed. For example, oncethe viral glycoprotein is modified, it can be used to prepare arecombinant virus by co-transfection with a viral vector containing areporter gene and at least one plasmid encoding virus packagingcomponents into a packaging cell line. The glycoprotein is incorporatedinto the viral envelope during budding of the virus. The virus can beused to transfect both a pure population of DCs as well as a mixedpopulation of cells containing DCs, and specificity of the viraltransduction of DCs can be confirmed by assaying the cells forexpression of the reporter gene in DCs and not to a significant extentin other cell types. If the specificity is not sufficiently stringent(for example, if undesired levels of infection of other cell types isobserved), the viral glycoprotein can be modified further and assayed asdescribed until the desired specificity is achieved.

Embodiments of the present invention include the delivery to DCs of DCactivators and/or maturation factors in conjunction with antigens.Exemplary DC activators and maturation factors include, but are notlimited to, stimulation molecules, cytokines, chemokines, antibodies andother agents such as Flt-3 ligands. For example, the DC maturationfactors can include at least one of the following: GM-CSF, IL-2, IL-4,IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand(CD4OL) and drug-inducible CD40 (iCD40) (Hanks, B. A., et al. 2005. NatMed 11:130-137, which is incorporated herein by reference in itsentirety).

Embodiments of the present invention also include methods andcompositions related to administration of recombinant virus as describedabove, or DCs infected with recombinant virus, into patients tostimulate antigen-specific immune responses, such as, for example, Tcell responses (cellular immune responses) and B cell responses (humoralimmune responses). For example, activated CD4 T cells can coordinate andorchestrate the CD8⁺ cytotoxic T cells and the B cells in anantigen-specific response. In preferred embodiments, the recombinantvirus and/or DCs infected with recombinant virus are used to stimulateimmune responses for the prevention and treatment of diseases such as,but not limited to, cancer and AIDS/HIV. Any disease can be treated forwhich an immune response to a particular antigen is beneficial,including, but not limited to, neoplastic disease, infectious disease,and immune-related diseases.

As herein described, studies were conducted that resulted in thediscovery of methods and compositions that can be used to directrecombinant viruses to provide genes encoding particular antigens intoDCs. The genetic modification of DCs in order to elicit productiveimmune responses can be used in the prevention and treatment of diseasesand provides an effective method of inducing effective T cell immunityas well as strong antibody production. The methods and compositionsdescribed herein can provide potent means for immunization with desiredantigens. Such immunization can prevent and treat diseases such as, forexample, cancer and AIDS/HIV.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g., Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N. Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N. Y.1989). Any methods, devices and materials similar or equivalent to thosedescribed herein can be used in the practice of this invention.

As used herein, the terms nucleic acid, polynucleotide and nucleotideare interchangeable and refer to any nucleic acid, whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sultone linkages, andcombinations of such linkages.

The terms nucleic acid, polynucleotide and nucleotide also specificallyinclude nucleic acids composed of bases other than the five biologicallyoccurring bases (adenine, guanine, thymine, cytosine and uracil).

As used herein, a nucleic acid molecule is said to be “isolated” whenthe nucleic acid molecule is substantially separated from contaminantnucleic acid molecules encoding other polypeptides.

“Immunization” refers to the provision of antigen to a host. In someembodiments, antigen is provided to antigen-presenting cells, such asdendritic cells. As described below, recombinant virus comprising a geneencoding an antigen can be targeted to dendritic cells with an affinitymolecule specific to DC-SIGN on dendritic cells. Thus the antigen towhich an immune response is desired can be delivered to the dendriticcells. Other methods of immunization are well known in the art.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), byantigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al.,J. Immunol. 156, 3901-3910) or by cytokine secretion. The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating IgG and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a patient,optionally in conjunction with an adjuvant.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments, enhances, and/or boosts the immuneresponse to the antigen, but when administered alone does not generatean immune response to the antigen. An adjuvant can be administered withthe recombinant virus of the invention as a single composition, or canbe administered before, concurrent with or after administration of therecombinant virus of the invention. Adjuvants can enhance an immuneresponse by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

The term “antibody” is used in the broadest sense and specificallycovers human, non-human (e.g. murine), chimeric, and humanizedmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), single-chain antibodies, and antibody fragments so long asthey exhibit the desired biological activity. Typically, fragmentscompete with the intact antibody from which they were derived forspecific binding to an antigen.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(see Burke, supra; Tigges, supra).

“Target cells” are any cells to which delivery of a polynucleotide or inwhich expression of a gene of interest is desired. Preferably, targetcells are dendritic cells, particularly dendritic cells that expressDC-SIGN.

The term “mammal” is defined as an individual belonging to the classMammalia and includes, without limitation, humans, domestic and farmanimals, and zoo, sports, and pet animals, such as sheep, dogs, horses,cats and cows.

The term “subject” or “patient” includes human and other mammaliansubjects that receive either prophylactic or therapeutic treatment.

As used herein, “treatment” is a clinical intervention that may betherapeutic or prophylactic. In therapeutic applications, pharmaceuticalcompositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. In prophylactic applications, pharmaceutical compositionsor medicants are administered to a patient susceptible to, or otherwiseat risk of, a particular disease in an amount sufficient to eliminate orreduce the risk or delay the outset of the disease. An amount adequateto accomplish this is defined as a therapeutically- orpharmaceutically-effective dose. Such an amount can be administered as asingle dosage or can be administered according to a regimen, whereby itis effective. The amount can cure a disease but, typically, isadministered in order to ameliorate the symptoms of a disease, or toeffect prophylaxis of a disease or disorder from developing. In boththerapeutic and prophylactic regimes, agents are usually administered inseveral dosages until a sufficient immune response has been achieved.Typically, the immune response is monitored and repeated dosages aregiven if the immune response starts to fade. “Treatment” need notcompletely eliminate a disease, nor need it completely prevent a subjectfrom becoming ill with the disease or disorder.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The term “cancer” refers to a disease or disorder that is characterizedby unregulated cell growth. Examples of cancer include, but are notlimited to, carcinoma, lymphoma, blastoma and sarcoma. Examples ofspecific cancers include, but are not limited to, lung cancer, coloncancer, breast cancer, testicular cancer, stomach cancer, pancreaticcancer, ovarian cancer, liver cancer, bladder cancer, colorectal cancer,and prostate cancer. Additional cancers are well known to those of skillin the art and include, but are not limited to: leukemia, lymphoma,cervical cancer, glioma tumors, adenocarcinomas and skin cancer.Exemplary cancers include, but are not limited to, a bladder tumor,breast tumor, prostate tumor, basal cell carcinoma, biliary tractcancer, bladder cancer, bone cancer, brain and CNS cancer (e.g., gliomatumor), cervical cancer, choriocarcinoma, colon and rectum cancer,connective tissue cancer, cancer of the digestive system; endometrialcancer, esophageal cancer; eye cancer; cancer of the head and neck;gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g. small cell and non-smallcell); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma;melanoma; myeloma, neuroblastoma, oral cavity cancer (e.g., lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer, retinoblastoma;rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratorysystem; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroidcancer; uterine cancer, cancer of the urinary system, as well as othercarcinomas and sarcomas. Cancer also includes neoplasias and malignantdisorders in mammals that are well known in the art.

A “vector” is a nucleic acid that is capable of transporting anothernucleic acid. Vectors may be, for example, plasmids, cosmids or phage.An “expression vector” is a vector that is capable of directingexpression of a protein or proteins encoded by one or more genes carriedby the vector when it is present in the appropriate environment.

The term “regulatory element” and “expression control element” are usedinterchangeably and refer to nucleic acid molecules that can influencethe transcription and/or translation of an operably linked codingsequence in a particular environment. These terms are used broadly andcover all elements that promote or regulate transcription, includingpromoters, core elements required for basic interaction of RNApolymerase and transcription factors, upstream elements, enhancers, andresponse elements (see, e.g., Lewin, “Genes V” (Oxford University Press,Oxford) pages 847-873). Exemplary regulatory elements in prokaryotesinclude promoters, operator sequences and a ribosome binding sites.Regulatory elements that are used in eukaryotic cells may include,without limitation, promoters, enhancers, splicing signals andpolyadenylation signals.

The term “transfection” refers to the introduction of a nucleic acidinto a host cell.

“Retroviruses” are viruses having an RNA genome.

“Lentivirus” refers to a genus of retroviruses that are capable ofinfecting dividing and non-dividing cells. Several examples oflentiviruses include HIV (human immunodeficiency virus: including HIVtype 1, and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates.

A lentiviral genome is generally organized into a 5′ long terminalrepeat (LTR), the gag gene, the pol gene, the env gene, the accessorygenes (nef, vif, vpr, vpu) and a 3′ LTR. The viral LTR is divided intothree regions called U3, R and U5. The U3 region contains the enhancerand promoter elements. The U5 region contains the polyadenylationsignals. The R (repeat) region separates the U3 and U5 regions andtranscribed sequences of the R region appear at both the 5′ and 3′ endsof the viral RNA. See, for example, “RNA Viruses: A Practical Approach”(Alan J. Cann, Ed., Oxford University Press, (2000)), 0 Narayan andClements J. Gen. Virology 70:1617-1639 (1989), Fields et al. FundamentalVirology Raven Press. (1990), Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma I M. J Virol. 72(10):8150-7 (1998), and U.S. Pat. No.6,013,516.

“Gammaretrovirus” refers to a genus of the retroviridae family.Exemplary gammaretroviruses include, but are not limited to, mouse stemcell virus, murine leukemia virus, feline leukemia virus, feline sarcomavirus, and avian reticuloendotheliosis viruses.

A “hybrid virus” as used herein refers to a virus having components fromone or more other viral vectors, including element from non-retroviralvectors, for example, adenoviral-retroviral hybrids. As used hereinhybrid vectors having a retroviral component are to be considered withinthe scope of the retroviruses.

“Virion,” “viral particle” and “retroviral particle” are used herein torefer to a single virus comprising an RNA genome, pol gene derivedproteins, gag gene derived proteins and a lipid bilayer displaying anenvelope (glyco)protein. The RNA genome is usually a recombinant RNAgenome and thus may contain an RNA sequence that is exogenous to thenative viral genome. The RNA genome may also comprise a defectiveendogenous viral sequence.

A “pseudotyped” retrovirus is a retroviral particle having an envelopeprotein that is from a virus other than the virus from which the RNAgenome is derived. The envelope protein can be, for example and withoutlimitation, from a different retrovirus or from a non-retroviral origin.The envelope protein can be a native envelope protein or an envelopeprotein that is modified, mutated or engineered as described herein. Insome embodiments, an envelope protein is a DC-SIGN-specific viralglycoprotein that is derived from a glycoprotein from one of thefollowing: Sindbis virus, influenza virus, Lassa fever virus, tick-borneencephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus,Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus,Hantaan virus, and SARS-CoV virus.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters a target cell. Transformation may rely on any knownmethod for the insertion of foreign nucleic acid sequences into aprokaryotic or eukaryotic host cell and may include, but is not limitedto, viral infection, electroporation, heat shock, lipofection, andparticle bombardment. “Transformed” cells include stably transformedcells in which the inserted nucleic acid is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome. Also included are cells that transiently express a gene ofinterest.

A “fusogenic molecule,” as described herein, is any molecule that cantrigger membrane fusion when present on the surface of a virus andallows a virus core to pass through the membrane and, typically, enterthe cytosol of a target cell. Fusogenic molecules can be, for example,viral glycoproteins. Exemplary viral glycoproteins contemplated asfusogenic molecules include, but are not limited to hemagglutinin,mutant hemagglutinin, SIN and viral glycoproteins from the followingviruses: Sindbis virus, influenza virus, Lassa fever virus, tick-borneencephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus,Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus,Hantaan virus, and SARS-CoV virus. Glycoproteins can be native ormodified to have desired activity.

By “transgene” is meant any nucleotide sequence, particularly a DNAsequence, that is integrated into one or more chromosomes of a host cellby human intervention, such as by the methods of the present invention.The transgene preferably comprises a “gene of interest.”

A “gene of interest” is not limited in any way and may be any nucleicacid, without limitation, that is desired to be delivered to,integrated, transcribed, translated, and/or expressed in a target cell.The gene of interest may encode a functional product, such as a proteinor an RNA molecule. Preferably the gene of interest encodes a protein orother molecule, the expression of which is desired in the target cell.The gene of interest is generally operatively linked to other sequencesthat are useful for obtaining the desired expression of the gene ofinterest, such as transcriptional regulatory sequences. In someembodiments a gene of interest is preferably one that encodes an antigento which an immune response is desired. Other genes of interest that maybe used in some embodiments are genes that encode dendritic cellactivators and/or maturation factors.

A “functional relationship” and “operably linked” mean, with respect tothe gene of interest, that the gene is in the correct location andorientation with respect to the promoter and/or enhancer that expressionof the gene will be affected when the promoter and/or enhancer iscontacted with the appropriate molecules.

“2A sequences” or elements are small peptides introduced as a linkerbetween two proteins, allowing autonomous intraribosomal self-processingof polyproteins (de Felipe. Genetic Vaccines and Ther. 2:13 (2004);deFelipe et al. Traffic 5:616-626 (2004)). The short peptides allowco-expression of multiple proteins from a single vector, such asco-expression of a fusogenic molecule and affinity molecule from thesame vector. Thus, in some embodiments polynucleotides encoding the 2Aelements are incorporated into a vector between polynucleotides encodingproteins to be expressed.

“DC maturation factors” (also known as “DC activators”) are compoundsthat can induce activation or stimulation of DCs such that DCsfacilitate the elicitation of cellular and humoral immune responses.Typical DC maturation factors are known in the art and include, but arenot limited to, stimulation molecules, cytokines, chemokines, antibodiesand other agents such as Flt-3 ligands (Figdor, C. G., et al. 2004. NatMed 10:475-480; Pulendran, B., et al. 2000. J Immunol 165: 566-572;Maraskovsky, E., et al. 2000. Blood 96:878-884, each of which isincorporated herein by reference in its entirety). Exemplary DCmaturation factors can include, but are not limited to, GM-CSF, IL-2,IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40ligand (CD4OL) and drug-inducible CD40 (iCD40).

Targeting Molecules

As discussed above, a targeting molecule is incorporated into arecombinant virus to target the virus to dendritic cells that expressDC-SIGN. The targeting molecule preferably also mediates fusion with thecell membrane and efficient transduction and delivery of the desiredpolynucleotide(s) into the dendritic cell. Thus, the targeting moleculeis typically a fusogenic molecule (FM) with the desired bindingspecificity. The targeting molecule is modified, if necessary, such thatit binds to DC-SIGN on dendritic cells. In some embodiments, thetargeting molecule specifically binds to DC-SIGN. That is, the targetingmolecule preferentially directs the recombinant virus to dendritic cellsthat express DC-SIGN relative to other cell types. Thus, in someembodiments, targeting molecules are created by eliminating the abilityof a FM to bind to other targets, such as hemagglutinin, while retainingthe ability to bind DC-SIGN. In other embodiments, the targetingmolecule can be modified to eliminate native binding specificity tonon-DC-SIGN molecules and components thereof and add or improve bindingspecificity for DC-SIGN. While some nonspecific binding to othermolecules, and thus other cell types, may occur even if the targetingmolecule is specific for DC-SIGN, the targeting molecules are modifiedto have sufficient specificity to avoid undesired side effects, such asside effects that may reduce the desired immune response.

Targeting molecules are generally molecules that are able to pseudotypevirus and thus be incorporated in the envelope of recombinant viruses,target dendritic cells and, under the right conditions, induce membranefusion and allow entry of a gene of interest to the dendritic cells.Preferred targeting molecules are viral glycoproteins. In addition,targeting molecules are preferably resistant to ultracentrifugation toallow concentration, which can be important for in vivo gene delivery.

Targeting molecules preferably induce membrane fusion at a low pH,independently of binding. Thus, in preferred embodiments, targetingmolecule-induced membrane fusion occurs once the virus comprising thetargeting molecule is inside the endosome of a target cell and the viralcore component, including a polynucleotide of interest, is delivered tothe cytosol.

In some embodiments a tag sequence is incorporated into a targetingmolecule to allow detection of targeting molecule expression and thepresence of the targeting molecule in viral particles.

There are two recognized classes of viral fusogens and both can be usedas targeting molecules (D. S. Dimitrov, Nature Rev. Microbio. 2, 109(2004)). The class I fusogens trigger membrane fusion using helicalcoiled-coil structures whereas the class II fusogens trigger fusion with13 barrels. These two structures have different mechanics and kinetics(D. S. Dimitrov, Nature Rev. Microbio. 2, 109 (2004)). In someembodiments, class I fusogens are used. In other embodiments, class IIfusogens are used. In still other embodiments, both class I and class IIfusogens are used.

Some non-limiting examples of surface glycoproteins that may be used astargeting molecules (or as fusogenic molecules in embodiments where theviral binding and fusion functions are separate), either in the wildtype or in modified form, include glycoproteins from alphaviruses, suchas Semliki Forest virus (SFV), Ross River virus (RRV) and Aura virus(AV), which comprise surface glycoproteins such as E1, E2, and E3. TheE2 glycoproteins derived from the Sindbis virus (SIN) and thehemagglutinin (HA) of influenza virus are non-retroviral glycoproteinsthat specifically bind particular molecules on cell surfaces (heparinsulfate glycosaminoglycan for E2, sialic acid for HA) and can be used tocreate targeting molecules in some embodiments. Their fusion isrelatively independent of binding to receptor molecules, and theactivation of fusion is accomplished through acidification in theendosome (Skehel and Wiley, Annu. Rev. Biochem. 69, 531-569 (2000);Smit, J. et al. J. Virol. 73, 8476-8484 (1999)). Moreover, they cantolerate certain genetic modifications and remain efficiently assembledon the retroviral surface (Morizono et al. J. Virol. 75, 8016-8020).

In other embodiments of the invention, surface glycoproteins of Lassafever virus, Hepatitis B virus, Rabies virus, Boma disease virus,Hantaan virus, or SARS-CoV virus can be utilized as fusion molecules.

In other embodiments of the invention, flavivirus-based surfaceglycoproteins may be used as the basis for targeting molecules. Likealphaviruses, flaviviruses use the class II fusion molecule to mediateinfection (Mukhopadhyay et al. (2005) Rev. Microbio. 3, 13-22). prM(about 165 amino acids) and E (about 495 amino acids) are theglycoproteins of flaviviruses. Also, the ligand-binding pocket for oneflavivirus, Dengue virus (DV), has been well-characterized. Of interest,DC-SIGN has been suggested to specifically interact with thecarbohydrate residues on the DV E protein to enhance viral entry(Mukhopadhyay et al. (2005) Nat. Rev. Microbio. 3, 13-22). Thus,lentiviruses enveloped only by DV E protein, or by modified DV Eprotein, can be used to target DCs. The TBE and DV E proteins, as wellas other fusion molecules described, may be engineered to provide thedesired binding specificity or to be binding deficient and fusioncompetent if necessary.

In some embodiments, a form of hemagglutinin (HA) from influenza A/fowlplague virus/Rostock/34 (FPV), a class I fusogen, is used (T.Hatziioannou, S. Valsesia-Wittmann, S. J. Russell, F. L. Cosset, J.Virol. 72, 5313 (1998)). In some embodiments, a form of FPV HA is used(A. H. Lin et al., Hum. Gene. Ther. 12, 323 (2001)). HA-mediated fusionis generally considered to be independent of receptor binding (D.Lavillette, S. J. Russell, F. L. Cosset, Curr. Opin. Biotech. 12, 461(2001)).

In other embodiments, a class II FM is used, preferably the Sindbisvirus glycoprotein from the alphavirus family (K. S. Wang, R. J. Kuhn,E. G. Strauss, S. Ou, J. H. Strauss, J. Virol. 66, 4992 (1992)), hereinalso referred to as SVG. SVG includes two transmembrane proteins (S.Mukhopadhyay, R. J. Kuhn, M. G. Rossmann, Nature Rev. Microbio. 3, 13(2005)), a first protein responsible for fusion (E1), and a secondprotein for cell binding (E2). SVG is known to pseudotype bothoncoretroviruses and lentiviruses.

As discussed below, in some preferred embodiments a modified SVG thatpreferentially binds DC-SIGN is utilized. In other embodiments, abinding-deficient and fusion-competent SVG, SVGmu, can be used as thefusogenic molecule in combination with a separate targeting molecule,such as an antibody to DC-SIGN or another dendritic cell specificprotein. For example, a SVG fusogenic molecule can be used in which theimmunoglobulin G binding domain of protein A (ZZ domain) is incorporatedinto the E2 protein and one or more additional mutations are made toinactivate the receptor binding sites (K. Morizono et al., Nature Med.11, 346 (2005)).

The gene encoding the targeting molecule is preferably cloned into anexpression vector, such as pcDNA3 (Invitrogen). Packaging cells, such as293T cells are then co-transfected with the viral vector encoding a geneof interest (typically encoding an antigen), at least one plasmidencoding virus packing components, and a vector for expression of thetargeting molecule. If the targeting function is separated from thefusogenic function, one or more vectors encoding an affinity moleculeand any associated components is also provided, The targeting moleculeis expressed on the membrane of the packaging cell and incorporated intothe recombinant virus. Expression of targeting molecules on thepackaging cell surface can be analyzed, for example, by FACS.

Based on information obtained, for example from structural studies andmolecular modeling, mutagenesis may be employed to generate the mutantforms of glycoproteins that maintain their fusogenic ability but havethe desired binding specificity and/or level of binding. Several mutantsmay be created for each glycoprotein and assayed using the methodsdescribed below, or other methods known in the art, to identify FMs withthe most desirable characteristics. For example, targeting molecules canbe tested for the ability to specifically deliver antigens to dendriticcells by determining their ability to stimulate an immune responsewithout causing undesired side effects in a mammal. The ability tospecifically target dendritic cells can also be tested directly, forexample, in cell culture as described below.

To select suitable targeting molecules (either wild-type or mutant),viruses bearing the targeting molecule (and an affinity molecule whereappropriate) are prepared and tested for their selectivity and/or theirability to facilitate penetration of the target cell membrane. Virusesthat display a wild-type glycoprotein can be used as controls forexamining titer effects in mutants. Cells expressing the binding partnerof the targeting molecule (or affinity molecule, where appropriate) aretransduced by the virus using a standard infection assay. After aspecified time, for example 48 hours post-transduction, cells can becollected and the percentage of cells infected by the virus comprisingthe targeting molecule (or affinity molecule and fusogenic molecule) canbe determined by, for example, FACS. The selectivity can be scored bycalculating the percentage of cells infected by virus. Similarly, theeffect of mutations on viral titer can be quantified by dividing thepercentage of cells infected by virus comprising a mutant targetingmolecule by the percentage of cells infected by virus comprising thecorresponding wild type targeting molecule. A preferred mutant will givethe best combination of selectivity and infectious titer. Once antargeting molecule is selected, viral concentration assays may beperformed to confinn that viruses enveloped by the FM can beconcentrated. Viral supernatants are collected and concentrated byultracentrifugation. The titers of viruses can be determined by limiteddilution of viral stock solution and transduction of cells expressingthe binding partner of the affinity molecule.

In some embodiments, BlaM-Vpr fusion protein may be utilized to evaluateviral penetration, and thus the efficacy of a fusion molecule (wild-typeor mutant). Virus may be prepared, for example, by transienttransfection of packaging cells with one or more vectors comprising theviral elements, BlaM-Vpr, and the FM of interest (and an affinitymolecule if appropriate). The resulting viruses can be used to infectcells expressing a molecule the targeting molecule (or affinitymolecule) specifically binds in the absence or presence of the freeinhibitor of binding (such as an antibody). Cells can then be washedwith CO₂-independent medium and loaded with CCF2 dye (AuroraBioscience). After incubation at room temperature to allow completion ofthe cleavage reaction, the cells can be fixed by paraformaldehyde andanalyzed by FACS and microscopy. The presence of blue cells indicatesthe penetration of viruses into the cytoplasm; fewer blue cells would beexpected when blocking antibody is added.

To investigate whether penetration is dependent upon a low pH, andselect targeting molecules (or fusogenic molecules) with the desired pHdependence, NH₄Cl or other compound that alters pH can be added at theinfection step (NH₄Cl will neutralize the acidic compartments ofendosomes). In the case of NH₄Cl, the disappearance of blue cells willindicate that penetration of viruses is low pH-dependent.

In addition, to confirm that the activity is pH-dependent,lysosomotropic agents, such as ammonium chloride, chloroquine,concanamycin, bafilomycin Al, monensin, nigericin, etc., may be addedinto the incubation buffer. These agents can elevate the pH within theendosomal compartments (e.g., Drose and Altendorf, J. Exp. Biol. 200,1-8, 1997). The inhibitory effect of these agents will reveal the roleof pH for viral fusion and entry. The different entry kinetics betweenviruses displaying different fusogenic molecules may be compared and themost suitable selected for a particular application.

PCR entry assays may be utilized to monitor reverse transcription andthus measure kinetics of viral DNA synthesis as an indication of thekinetics of viral entry. For example, viral particles comprising aparticular targeting molecule may be incubated with packaging cells,such as 293T cells, expressing the appropriate cognate for the targetingmolecule (or a separate affinity molecule in some embodiments). Eitherimmediately, or after incubation (to allow infection to occur) unboundviruses are removed and aliquots of the cells are analyzed. DNA may thenbe extracted from these aliquots and semi-quantitative performed usingLTR-specific primers. The appearance of LTR-specific DNA products willindicate the success of viral entry and uncoating.

Although the targeting molecule can have both viral binding and fusionfunctions, in another aspect of the invention, the viral binding andfusion functions are separated into two distinct components. Typically,the recombinant virus comprises both (i) an affinity molecule thatmediates viral binding and precisely targets the virus to dendriticcells, and (ii) a distinct fusogenic molecule (FM) that mediatesefficient transduction and delivery of the desired polynucleotide intothe dendritic cells. The methods disclosed herein may be readily adoptedto utilize any of a variety of affinity molecules and fusogenicmolecules. In addition to those described herein, other exemplaryfusogenic molecules and related methods are described, for example, inU.S. patent application Ser. No. 11/071,785, filed Mar. 2, 2005(published as U.S. Patent Application Publication 2005-0238626), and inU.S. patent application Ser. No. 11/446,353, filed Jun. 1, 2006(published as U.S. Patent Application Publication 2007/0020238), each ofwhich is incorporated herein by reference in its entirety.

The affinity molecule is one that binds a dendritic cell surface marker.In preferred embodiments, the affinity molecule binds DC-SIGN withspecificity. That is, the binding of the affinity molecule to DC-SIGN ispreferably specific enough to avoid undesired side effects due tointeraction with markers on other cell types. The affinity molecule canbe, for example, an antibody that specifically binds DC-SIGN.

In some preferred embodiments, the fusion molecule is a viralglycoprotein that mediates fusion or otherwise facilitates delivery ofthe gene of interest to the dendritic cell, preferably in response tothe low pH environment of the endosome. The fusion molecule preferablyexhibits fast enough kinetics that the viral contents can empty into thecytosol before the degradation of the viral particle. In addition, thefusion molecule can be modified to reduce or eliminate any bindingactivity and thus reduce or eliminate any non-specific binding. That is,by reducing the binding ability of the fusion molecules, binding of thevirus to the target cell is determined predominantly or entirely by theaffinity molecule, allowing for high target specificity and reducingundesired effects. Exemplary fusion molecules include, but are notlimited to viral glycoproteins derived from one of the followingviruses: Sindbis virus, influenza virus, Lassa fever virus, tick-borneencephalitis virus, Dengue virus, Hepatitis B virus, Rabies virus,Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus,Hantaan virus, and SARS-CoV virus.

The methods disclosed herein can be readily adopted to utilize any of avariety of molecules as targeting molecules, or as fusogenic moleculesin combination with affinity molecules. In addition to those describedherein, other exemplary molecules and related methods are described, forexample, in U.S. Patent Application Publication 2005/0238626 and in U.S.Patent Application Publication 2007/0020238).

Vectors

In a preferred embodiment, one or more vectors are used to introducepolynucleotide sequences into a packaging cell line for the preparationof a recombinant virus as described herein. The vectors can containpolynucleotide sequences encoding the various components of therecombinant virus including the DC-specific targeting molecule, agene(s) of interest (typically encoding an antigen), and any componentsnecessary for the production of the virus that are not provided by thepackaging cell. In some embodiments, vectors containing polynucleotidesequences that encode a DC-specific affinity molecule and a separatefugosenic molecule are substituted for a vector that encodes aDC-specific targeting molecule in the preparation of the virus.Eukaryotic cell expression vectors are well known in the art and areavailable from a number of commercial sources.

In one aspect of the invention, vectors containing polynucleotidesequences that encode DC maturation factors are also used in thepreparation of the virus. These polynucleotides are typically under thecontrol of one or more regulatory elements that direct the expression ofthe coding sequences in the packaging cell and the target cell, asappropriate. Several lines of evidence have shown the success of DCvaccination is dependent on the maturation state of DCs (Banchereau, Jand Palucka, A. K. Nat. Rev. Immunol. 5:296-306 (2005); Schuler, G. etal. Curr. Opin. Immuno 1.15:138-147 (2003); Figdor, C. G. et al. Nat.Med. 10:475-480 (2004), each of which is incorporated herein byreference). Maturation can transform DCs from cells actively involved inantigen capture into cells specialized for T cell priming. In one aspectof the invention, the vector includes genes that encode the stimulatorymolecules to trigger the desired DC maturation. Such stimulatorymolecules are also referred to as maturation factors or maturationstimulatory factors.

In some embodiments, packaging cells are co-transfected with a viralvector encoding an antigen and one or more additional vectors. Forexample, in addition to the viral vector encoding an antigen, a secondvector preferably carries a gene encoding a targeting molecule thatbinds dendritic cells, such SVGmu, as described elsewhere in theapplication. In some preferred embodiments, the targeting moleculeencodes a modified viral glycoprotein that is specific for DC-SIGN. Themodified viral glycoprotein is preferably one derived from at least oneof the following: Sindbis virus, influenza virus, Lassa fever virus,tick-borne encephalitis virus, Dengue virus, Hepatitis B virus, Rabiesvirus, Semliki Forest virus, Ross River virus, Aura virus, Borna diseasevirus, Hantaan virus, and SARS-CoV virus. In some embodiments, the viralvector encoding an antigen also includes a polynucleotide sequenceencoding a DC maturation factor. In some embodiments, the polynucleotidesequence encoding a DC maturation factor is contained in a third vectorthat is co-transfected with the viral vector encoding an antigen and theone or more additional vectors into the packaging cells.

In other embodiments, one or more multicistronic expression vectors areutilized that include two or more of the elements (e.g., the viralgenes, gene(s) of interest, the targeting molecule, DC maturationfactors) necessary for production of the desired recombinant virus inpackaging cells. The use of multicistronic vectors reduces the totalnumber of vectors required and thus avoids the possible difficultiesassociated with coordinating expression from multiple vectors. In amulticistronic vector the various elements to be expressed are operablylinked to one or more promoters (and other expression control elementsas necessary). In other embodiments a multicistronic vector comprising agene of interest, a reporter gene, and viral elements is used. The geneof interest typically encodes an antigen and, optionally, a DCmaturation factor. Such a vector may be cotransfected, for example,along with a vector encoding a targeting molecule, or, in someembodiments, a multicistronic vector encoding both an FM and an affinitymolecule. In some embodiments the multicistronic vector comprises a geneencoding an antigen, a gene encoding a DC maturation factor and viralelements.

Each component to be expressed in a multicistronic expression vector maybe separated, for example, by an IRES element or a viral 2A element, toallow for separate expression of the various proteins from the samepromoter. IRES elements and 2A elements are known in the art (U.S. Pat.No. 4,937,190; de Felipe et al. 2004. Traffic 5: 616-626, each of whichis incorporated herein by reference in its entirety). In one embodiment,oligonucleotides encoding furin cleavage site sequences (RAKR) (Fang etal. 2005. Nat. Biotech 23: 584-590, which is incorporated herein byreference in its entirety) linked with 2A-like sequences fromfoot-and-mouth diseases virus (FMDV), equine rhinitis A virus (ERAV),and thosea asigna virus (TaV) (Szymczak et al. 2004. Nat. Biotechnol.22: 589-594, which is incorporated herein by reference in its entirety)are used to separate genetic elements in a multicistronic vector. Theefficacy of a particular multicistronic vector for use in synthesizingthe desired recombinant virus can readily be tested by detectingexpression of each of the genes using standard protocols.

Generation of the vector(s) can be accomplished using any suitablegenetic engineering techniques known in the art, including, withoutlimitation, the standard techniques of restriction endonucleasedigestion, ligation, transformation, plasmid purification, and DNAsequencing, for example as described in Sambrook et al. (1989. MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y.), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press,N. Y. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann,Ed., Oxford University Press, (2000), each of the foregoing which isincorporated herein by reference in its entirety.

The vector(s) may incorporate sequences from the genome of any knownorganism. The sequences may be incorporated in their native form or maybe modified in any way. For example, the sequences may compriseinsertions, deletions or substitutions.

Expression control elements that may be used for regulating theexpression of the components are known in the art and include, but arenot limited to, inducible promoters, constitutive promoters, secretionsignals, enhancers and other regulatory elements.

In one embodiment, a vector can include a prokaryotic replicon, i.e., aDNA sequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extrachromosomally in aprokaryotic host cell, such as a bacterial host cell, transformedtherewith. Such replicons are well known in the art. In addition,vectors that include a prokaryotic replicon may also include a genewhose expression confers a detectable marker such as a drug resistance.Typical bacterial drug resistance genes are those that confer resistanceto ampicillin or tetracycline.

The vector(s) may include one or more genes for selectable markers thatare effective in a eukaryotic cell, such as a gene for a drug resistanceselection marker. This gene encodes a factor necessary for the survivalor growth of transformed host cells grown in a selective culture medium.Host cells not transformed with the vector containing the selection genewill not survive in the culture medium. Typical selection genes encodeproteins that confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, complementauxotrophic deficiencies, or supply critical nutrients withheld from themedia. The selectable marker can optionally be present on a separateplasmid and introduced by co-transfection.

Vectors will usually contain a promoter that is recognized by thepackaging cell and that is operably linked to the polynucleotide(s)encoding the targeting molecule, viral components, and the like. Apromoter is an expression control element formed by a nucleic acidsequence that permits binding of RNA polymerase and transcription tooccur. Promoters are untranslated sequences that are located upstream(5′) to the start codon of a structural gene (generally within about 100to 1000 bp) and control the transcription and translation of theantigen-specific polynucleotide sequence to which they are operablylinked. Promoters may be inducible or constitutive. The activity of theinducible promoters is induced by the presence or absence of biotic orabiotic factors. Inducible promoters can be a useful tool in geneticengineering because the expression of genes to which they are operablylinked can be turned on or off at certain stages of development of anorganism or in a particular tissue. Inducible promoters can be groupedas chemically-regulated promoters, and physically-regulated promoters.Typical chemically-regulated promoters include, not are not limited to,alcohol-regulated promoters (e.g. alcohol dehydrogenase I (alcA) genepromoter), tetracycline-regulated promoters (e.g.tetracycline-responsive promoter), steroid-regulated promoter (e.g. ratglucocorticoid receptor (GR)-based promoter, human estrogen receptor(ER)-based promoter, moth ecdysone receptor-based promoter, and thepromoters based on the steroid/retinoid/thyroid receptor superfamily),metal-regulated promoters (e.g. metallothionein gene-based promoters),and pathogenesis-related promoters (e.g. Arabidopsis and maizepathogen-related (PR) protein-based promoters). Typicalphysically-regulated promoters include, but are not limited to,temperature-regulated promoters (e.g. heat shock promoters), andlight-regulated promoters (e.g. soybean SSU promoter). Other exemplarypromoters are described elsewhere, for example, in hyper text transferprotocol: //www.patentlens.net/daisy/promoters/768/271.html, which isincorporated herein by reference in its entirety.

One of skill in the art will be able to select an appropriate promoterbased on the specific circumstances. Many different promoters are wellknown in the art, as are methods for operably linking the promoter tothe gene to be expressed. Both native promoter sequences and manyheterologous promoters may be used to direct expression in the packagingcell and target cell. However, heterologous promoters are preferred, asthey generally permit greater transcription and higher yields of thedesired protein as compared to the native promoter.

The promoter may be obtained, for example, from the genomes of virusessuch as polyoma virus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40). The promoter may also be, for example,a heterologous mammalian promoter, e.g., the actin promoter or animmunoglobulin promoter, a heat-shock promoter, or the promoter normallyassociated with the native sequence, provided such promoters arecompatible with the target cell. In one embodiment, the promoter is thenaturally occurring viral promoter in a viral expression system. In someembodiments, the promoter is a dendritic cell-specific promoter. Thedendritic cell-specific promoter can be, for example, CD11c promoter.

Transcription may be increased by inserting an enhancer sequence intothe vector(s). Enhancers are typically cis-acting elements of DNA,usually about 10 to 300 bp in length, that act on a promoter to increaseits transcription. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, a-fetoprotein, and insulin).Preferably an enhancer from a eukaryotic cell virus will be used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. The enhancer may be spliced into the vector at aposition 5′ or 3′ to the antigen-specific polynucleotide sequence, butis preferably located at a site 5′ from the promoter.

Expression vectors will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Thesesequences are often found in the 5′ and, occasionally 3′, untranslatedregions of eukaryotic or viral DNAs or cDNAs and are well known in theart.

Plasmid vectors containing one or more of the components described aboveare readily constructed using standard techniques well known in the art.

For analysis to confirm correct sequences in plasmids constructed, theplasmid may be replicated in E. coli, purified, and analyzed byrestriction endonuclease digestion, and/or sequenced by conventionalmethods.

Vectors that provide for transient expression in mammalian cells mayalso be used. Transient expression involves the use of an expressionvector that is able to replicate efficiently in a host cell, such thatthe host cell accumulates many copies of the expression vector and, inturn, synthesizes high levels of a the polypeptide encoded by theantigen-specific polynucleotide in the expression vector. See Sambrooket al., supra, pp. 16.17-16.22.

Other vectors and methods suitable for adaptation to the expression ofthe viral polypeptides are well known in the art and are readily adaptedto the specific circumstances.

Using the teachings provided herein, one of skill in the art willrecognize that the efficacy of a particular expression system can betested by transforming packaging cells with a vector comprising a geneencoding a reporter protein and measuring the expression using asuitable technique, for example, measuring fluorescence from a greenfluorescent protein conjugate. Suitable reporter genes are well known inthe art.

Transformation of packaging cells with vectors of the present inventionis accomplished by well-known methods, and the method to be used is notlimited in any way. A number of non-viral delivery systems are known inthe art, including for example, electroporation, lipid-based deliverysystems including liposomes, delivery of “naked” DNA, and delivery usingpolycyclodextrin compounds, such as those described in Schatzlein A G.(2001. Non-Viral Vectors in Cancer Gene Therapy: Principles andProgresses. Anticancer Drugs, which is incorporated herein by referencein its entirety). Cationic lipid or salt treatment methods are typicallyemployed, see, for example, Graham et al. (1973. Virol. 52:456; Wigleret al. (1979. Proc. Natl. Acad. Sci. USA 76:1373-76), each of theforegoing which is incorporated herein by reference in its entirety. Thecalcium phosphate precipitation method is preferred. However, othermethods for introducing the vector into cells may also be used,including nuclear microinjection and bacterial protoplast fusion.

Viral Vector and Packaging Cells

One of the vectors encodes the core virus (the “viral vector”). Thereare a large number of available viral vectors that are suitable for usewith the invention, including those identified for human gene therapyapplications, such as those described by Pfeifer and Verma (Pfeifer, A.and I. M. Verma. 2001. Annu. Rev. Genomics Hum. Genet. 2:177-211, whichis incorporated herein by reference in its entirety). Suitable viralvectors include vectors based on RNA viruses, such as retrovirus-derivedvectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, andinclude more complex retrovirus-derived vectors, e.g.,lentivirus-derived vectors. Human Immunodeficiency virus (HIV-1)-derivedvectors belong to this category. Other examples include lentivirusvectors derived from HIV-2, feline immunodeficiency virus (FIV), equineinfectious anemia virus, simian immunodeficiency virus (SIV) andmaedi/visna virus.

The viral vector preferably comprises one or more genes encodingcomponents of the recombinant virus as well as one or more genes ofinterest, such as, for example, an antigen and/or a DC maturationfactor. The viral vector may also comprise genetic elements thatfacilitate expression of the gene of interest in a target cell, such aspromoter and enhancer sequences. In order to prevent replication in thetarget cell, endogenous viral genes required for replication may beremoved and provided separately in the packaging cell line.

In a preferred embodiment the viral vector comprises an intactretroviral 5′ LTR and a self-inactivating 3′ LTR.

Any method known in the art may be used to produce infectious retroviralparticles whose genome comprises an RNA copy of the viral vector. Tothis end, the viral vector (along with other vectors encoding the geneof interest, the DC-specific targeting molecule, etc.) is preferablyintroduced into a packaging cell line that packages viral genomic RNAbased on the viral vector into viral particles.

The packaging cell line provides the viral proteins that are required intrans for the packaging of the viral genomic RNA into viral particles.The packaging cell line may be any cell line that is capable ofexpressing retroviral proteins. Preferred packaging cell lines include293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The packaging cellline may stably express the necessary viral proteins. Such a packagingcell line is described, for example, in U.S. Pat. No. 6,218,181, whichis incorporated herein by reference in its entirety. Alternatively apackaging cell line may be transiently transfected with plasmidscomprising nucleic acid that encodes one or more necessary viralproteins, including the DC-specific targeting molecule (oralternatively, a DC-specific affinity molecule and fusogenic molecule)along with the viral vectors encoding the gene of interest, whichtypically encodes an antigen and can additionally encode a DC maturationfactor.

Viral particles comprising a polynucleotide with the gene of interestand a targeting molecule that is specific for dendritic cells arecollected and allowed to infect the target cell. In some preferredembodiments, the virus is pseudotyped to achieve target cellspecificity. Methods for pseudotyping are well known in the art and alsodescribed herein.

In one embodiment, the recombinant virus used to deliver the gene ofinterest is a modified lentivirus and the viral vector is based on alentivirus. As lentiviruses are able to infect both dividing andnon-dividing cells, in this embodiment it is not necessary for targetcells to be dividing (or to stimulate the target cells to divide).

In another embodiment, the recombinant virus used to deliver the gene ofinterest is a modified gammaretrovirus and the viral vector is based ona gammaretrovirus.

In another embodiment the vector is based on the murine stem cell virus(MSCV; (Hawley, R. G., et al. (1996) Proc. Natl. Acad. Sci. USA93:10297-10302; Keller, G., et al. (1998) Blood 92:877-887; Hawley, R.G., et al. (1994) Gene Ther. 1:136-138, each of the foregoing which isincorporated herein by reference in its entirety). The MSCV vectorprovides long-term stable expression in target cells, particularlyhematopoietic precursor cells and their differentiated progeny.

In another embodiment, the vector is based on a modified Moloney virus,for example a Moloney Murine Leukemia Virus. The viral vector can alsocan be based on a hybrid virus such as that described in Choi, J. K., etal. (2001. Stem Cells 19, No. 3, 236-246, which is incorporated hereinby reference in its entirety).

A DNA viral vector may be used, including, for example adenovirus-basedvectors and adeno-associated virus (AAV)-based vectors. Likewise,retroviral-adenoviral vectors also can be used with the methods of theinvention.

Other vectors also can be used for polynucleotide delivery includingvectors derived from herpes simplex viruses (HSVs), including ampliconvectors, replication-defective HSV and attenuated HSV (Krisky D M,Marconi P C, Oligino T J, Rouse R J, Fink D J, et al. 1998. Developmentof herpes simplex virus replication-defective multigene vectors forcombination gene therapy applications. Gene Ther. 5: 1517-30, which isincorporated herein by reference in its entirety).

Other vectors that have recently been developed for gene therapy usescan also be used with the methods of the invention. Such vectors includethose derived from baculoviruses and alpha-viruses. (Jolly D J. 1999.Emerging viral vectors. pp 209-40 in Friedmann T, ed. 1999. Thedevelopment of human gene therapy. New York: Cold Spring Harbor Lab,which is incorporated herein by reference in its entirety).

In some preferred embodiments, the viral construct comprises sequencesfrom a lentivirus genome, such as the HIV genome or the SIV genome. Theviral construct preferably comprises sequences from the 5′ and 3′ LTRsof a lentivirus. More preferably the viral construct comprises the R andU5 sequences from the 5′ LTR of a lentivirus and an inactivated orself-inactivating 3′ LTR from a lentivirus. The LTR sequences may be LTRsequences from any lentivirus from any species. For example, they may beLTR sequences from HIV, SIV, FIV or BIV. Preferably the LTR sequencesare HIV LTR sequences.

The viral construct preferably comprises an inactivated orself-inactivating 3′ LTR. The 3′ LTR may be made self-inactivating byany method known in the art. In the preferred embodiment the U3 elementof the 3′ LTR contains a deletion of its enhancer sequence, preferablythe TATA box, Spl and NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

Optionally, the U3 sequence from the lentiviral 5′ LTR may be replacedwith a promoter sequence in the viral construct. This may increase thetiter of virus recovered from the packaging cell line. An enhancersequence may also be included. Any enhancer/promoter combination thatincreases expression of the viral RNA genome in the packaging cell linemay be used. In a preferred embodiment the CMV enhancer/promotersequence is used.

In some preferred embodiments, the viral construct comprises sequencesfrom a gammaretrovirus genome, such as the mouse stem cell virus (MSCV)genome or the murine leukemia virus (MLV) genome. The viral constructpreferably comprises sequences from the 5′ and 3′ LTRs of agammaretrovirus. The LTR sequences may be LTR sequences from anygammaretrovirus from any species. For example, they may be LTR sequencesfrom mouse stem cell virus (MSCV), murine leukemia virus (MLV), felineleukemia virus (FLV), feline sarcoma virus (FAV), and avianreticuloendotheliosis viruses (ARV). Preferably the LTR sequences areMSCV and MLV LTR sequences.

In some embodiments, the viral construct preferably comprises aninactivated or self-inactivating 3′ LTR. The 3′ LTR may be madeself-inactivating by any method known in the art. In the preferredembodiment the U3 element of the 3′ LTR contains a deletion of itsenhancer sequence, preferably the TATA box, Spl and NF-kappa B sites. Asa result of the self-inactivating 3′ LTR, the provirus that isintegrated into the host cell genome will comprise an inactivated 5′LTR.

Optionally, the U3 sequence from the gammaretroviral 5′ LTR may bereplaced with a promoter sequence in the viral construct. This mayincrease the titer of virus recovered from the packaging cell line. Anenhancer sequence may also be included. Any enhancer/promotercombination that increases expression of the viral RNA genome in thepackaging cell line may be used. In a preferred embodiment the CMVenhancer/promoter sequence is used.

The viral construct generally comprises a gene that encodes an antigenthat is desirably expressed in one or more target cells. Preferably thegene of interest is located between the 5′ LTR and 3′ LTR sequences.Further, the gene of interest is preferably in a functional relationshipwith other genetic elements, for example transcription regulatorysequences such as promoters and/or enhancers, to regulate expression ofthe gene of interest in a particular manner once the gene isincorporated into the target cell. In certain embodiments, the usefultranscriptional regulatory sequences are those that are highly regulatedwith respect to activity, both temporally and spatially.

In some embodiments, the gene of interest is in a functionalrelationship with internal promoter/enhancer regulatory sequences. An“internal” promoter/enhancer is one that is located between the 5′ LTRand the 3′ LTR sequences in the viral construct and is operably linkedto the gene that is desirably expressed.

The internal promoter/enhancer may be any promoter, enhancer orpromoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship. A “functionalrelationship” and “operably linked” mean, without limitation, that thegene is in the correct location and orientation with respect to thepromoter and/or enhancer that expression of the gene will be affectedwhen the promoter and/or enhancer is contacted with the appropriatemolecules.

The internal promoter/enhancer is preferably selected based on thedesired expression pattern of the gene of interest and the specificproperties of known promoters/enhancers. Thus, the internal promoter maybe a constitutive promoter. Non-limiting examples of constitutivepromoters that may be used include the promoter for ubiquitin, CMV(Karasuyama et al. 1989. J. Exp. Med. 169:13, which is incorporatedherein by reference in its entirety), beta-actin (Gunning et al. 1989.Proc. Natl. Acad. Sci. USA 84:4831-4835, which is incorporated herein byreference in its entirety) and pgk (see, for example, Adra et al. 1987.Gene 60:65-74; Singer-Sam et al. 1984. Gene 32:409-417; and Dobson etal. 1982. Nucleic Acids Res. 10:2635-2637, each of the foregoing whichis incorporated herein by reference in its entirety).

Alternatively, the promoter may be a tissue specific promoter. In somepreferred embodiments, the promoter is a target cell-specific promoter.For example, the promoter can be the dendritic cell-specific promoterCD11c (Masood, R., et al. 2001. Int J Mol Med 8:335-343; Somia, N. V.,et al. 1995. Proc Acad Sci USA 92:7570-7574, each of which isincorporated herein by reference in its entirety.) In addition,promoters may be selected to allow for inducible expression of the gene.A number of systems for inducible expression are known in the art,including the tetracycline responsive system and the lacoperator-repressor system. It is also contemplated that a combination ofpromoters may be used to obtain the desired expression of the gene ofinterest. The skilled artisan will be able to select a promoter based onthe desired expression pattern of the gene in the organism and/or thetarget cell of interest.

In some embodiments the viral construct preferably comprises at leastone RNA Polymerase II or III promoter. The RNA Polymerase II or IIIpromoter is operably linked to the gene of interest and can also belinked to a termination sequence. In addition, more than one RNAPolymerase II or III promoters may be incorporated.

RNA polymerase II and III promoters are well known to one of skill inthe art. A suitable range of RNA polymerase III promoters can be found,for example, in Paule and White. Nucleic Acids Research., Vol 28, pp1283-1298 (2000), which is incorporated herein by reference in itsentirety. The definition of RNA polymerase II or III promoters,respectively, also include any synthetic or engineered DNA fragment thatcan direct RNA polymerase II or III, respectively, to transcribe itsdownstream RNA coding sequences. Further, the RNA polymerase II or III(Pol II or III) promoter or promoters used as part of the viral vectorcan be inducible. Any suitable inducible Pol II or III promoter can beused with the methods of the invention. Particularly suited Pol II orIII promoters include the tetracycline responsive promoters provided inOhkawa and Taira Human Gene Therapy, Vol. 11, pp 577-585 (2000) and inMeissner et al. Nucleic Acids Research, Vol. 29, pp 1672-1682 (2001),each of which is incorporated herein by reference in its entirety.

An internal enhancer may also be present in the viral construct toincrease expression of the gene of interest. For example, the CMVenhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, which isincorporated herein by reference in its entirety) may be used. In someembodiments, the CMV enhancer can be used in combination with thechicken 13-actin promoter. One of skill in the art will be able toselect the appropriate enhancer based on the desired expression pattern.

The polynucleotide or gene of interest is not limited in any way andincludes any nucleic acid that the skilled practitioner desires to haveintegrated, transcribed, translated, and/or expressed in the targetcell. In some embodiments, the polynucleotide can be a gene that encodesan antigen against which an immune response is desired. In someembodiments, the polynucleotide can be a gene encoding a smallinhibiting RNA (siRNA) or a microRNA (miRNA) of interest thatdown-regulates expression of a molecule. For example, the gene encodingan siRNA or a microRNA can be used to down-regulate expression ofnegative regulators in a cell, including those that inhibit activationor maturation of dendritic cells. siRNAs and microRNAs are known in theart and describe elsewhere (Shen, L. et al. 2004. Nat Biotech 22(12):1546-1553; Zhou, H. et al. 2006. Biochemical and Biophysical ResearchCommunications 347:200-207; Song, X-T., et al. 2006. PLoS Medicine3(1):el 1; Kobayashi, T. and A. Yoshimura. 2005. TRENDS in Immunology26(4):177-179; Taganov, K., et al. 2007. Immunity 26:133-137; Dahlberg,J. E. and E. Lund. 2007. Sci. STKE 387:pe25, each of which isincorporated herein by reference in its entirety).

In addition, in some embodiments, the polynucleotide can contain morethan one gene of interest, which can be placed in functionalrelationship with the viral promoter. The gene of interest can encode aprotein, a siRNA, or a microRNA. In some embodiments, the polynucleotideto be delivered can comprise multiple genes encoding at least oneprotein, at least one siRNA, at least one microRNA, or any combinationsthereof. For example, the polynucleotide to be delivered can include oneor more genes that encode one or more antigens against which an immuneresponse is desired. The one or more antigens can be associated with asingle disease or disorder, or the can be associated with multiplediseases and/or disorders. In some embodiments, a gene encoding animmune regulatory protein can be constructed with a primary geneencoding an antigen against which an immune response is desired, and thecombination can elicit and regulate the immune response to the desireddirection and magnitude. In some embodiments, a gene encoding an siRNAor microRNA can be constructed with a primary gene encoding an antigenagainst which an immune response is desired, and the combination canregulate the scope of the immune response. (See, for example,embodiments of polynucleotides in FIG. 24c and FIG. 24d , withaccompanying sequences in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.)In some embodiments, a gene encoding a marker protein can be placedafter a primary gene of interest to allow for identification of cellsthat are expressing the desired protein. In one embodiment a fluorescentmarker protein, preferably green fluorescent protein (GFP), isincorporated into the construct along with the gene of interest(typically encoding an antigen). If more than one gene is included,internal ribosomal entry site (IRES) sequences, or 2A elements are alsopreferably included, separating the primary gene of interest from areporter gene and/or any other gene of interest. The IRES or 2Asequences may facilitate the expression of the reporter gene, or othergenes.

The viral construct may also contain additional genetic elements. Thetypes of elements that may be included in the construct are not limitedin any way and will be chosen by the skilled practitioner to achieve aparticular result. For example, a signal that facilitates nuclear entryof the viral genome in the target cell may be included. An example ofsuch a signal is the HIV-1 flap signal.

Further, elements may be included that facilitate the characterizationof the provirus integration site in the target cell. For example, a tRNAamber suppressor sequence may be included in the construct.

In addition, the construct may contain one or more genetic elementsdesigned to enhance expression of the gene of interest. For example, awoodchuck hepatitis virus responsive element (WRE) may be placed intothe construct (Zufferey et al. 1999. J. Virol. 74:3668-3681; Deglon etal. 2000. Hum. Gene Ther. 11:179-190, each of which is incorporatedherein by reference in its entirety).

A chicken 13-globin insulator may also be included in the viralconstruct. This element has been shown to reduce the chance of silencingthe integrated provirus in the target cell due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous gene from positive ornegative positional effects from surrounding DNA at the integration siteon the chromosome.

Any additional genetic elements are preferably inserted 3′ of the geneof interest.

In a specific embodiment, the viral vector comprises: a cytomegalovirus(CMV) enhancer/promoter sequence; the R and U5 sequences from the HIV 5′LTR; the HIV-1 flap signal; an internal enhancer; an internal promoter;a gene of interest; the woodchuck hepatitis virus responsive element; atRNA amber suppressor sequence; a U3 element with a deletion of itsenhancer sequence; the chicken beta-globin insulator; and the R and U5sequences of the 3′ HIV LTR.

The viral construct is preferably cloned into a plasmid that may betransfected into a packaging cell line. The preferred plasmid preferablycomprises sequences useful for replication of the plasmid in bacteria.

Delivery of the Virus

The virus may be delivered to a target cell in any way that allows thevirus to contact the target dendritic cells (DCs) in which delivery of apolynucleotide of interest is desired. In preferred embodiments, asuitable amount of virus is introduced into an animal directly (invivo), for example though injection into the body. In some preferredembodiments, the viral particles are injected into a mammal's peripheralblood stream. In other preferred embodiments, the viral particles areinjected into a mammal through intra-dermal injection, subcutaneousinjection, intra-peritoneal cavity injection, or intra-venal injection.The virus may be delivered using a subdermal injection device such thedevices disclosed in U.S. Pat. Nos. 7,241,275, 7,115,108, 7,108,679,7,083,599, 7,083,592, 7,047,070, 6,971,999, 6,808,506, 6,780,171,6,776,776, 6,689,118, 6,670,349, 6,569,143, 6,494,865, 5,997,501,5,848,991, 5,328,483, 5,279,552, 4,886,499, all of which areincorporated by reference in their entirety for all purposes. Otherinjection locations also are suitable, such as directly into organscomprising target cells. For example intra-lymph node injection,intra-spleen injection, or intra-bone marrow injection may be used todeliver virus to the lymph node, the spleen and the bone marrow,respectively. Depending on the particular circumstances and nature ofthe target cells, introduction can be carried out through other meansincluding for example, inhalation, or direct contact with epithelialtissues, for example those in the eye, mouth or skin.

In other embodiments, target cells are provided and contacted with thevirus in vitro, such as in culture plates. The target cells aretypically dendritic cells obtained from a healthy subject or a subjectin need of treatment. Preferably, the target cells are dendritic cellsobtained from a subject in whom it is desired to stimulate an immuneresponse to an antigen. Methods to obtain cells from a subject are wellknown in the art. The virus may be suspended in media and added to thewells of a culture plate, tube or other container. The media containingthe virus may be added prior to the plating of the cells or after thecells have been plated. Preferably cells are incubated in an appropriateamount of media to provide viability and to allow for suitableconcentrations of virus in the media such that infection of the hostcell occurs.

The cells are preferably incubated with the virus for a sufficientamount of time to allow the virus to infect the cells. Preferably thecells are incubated with virus for at least 1 hour, more preferably atleast 5 hours and even more preferably at least 10 hours.

In both in vivo and in vitro delivery embodiments, any concentration ofvirus that is sufficient to infect the desired target cells may be used,as can be readily determined by the skilled artisan. When the targetcell is to be cultured, the concentration of the viral particles is atleast 1 PFU/ul, more preferably at least 10 PFU/ul, even more preferablyat least 400 PFU/μ1 and even more preferably at least 1×10⁴ PFU/ul.

In some embodiments, following infection with the virus in vitro, targetcells can be introduced (or re-introduced) into an animal. In someembodiments, the cells can be introduced into the dermis, under thedermis, or into the peripheral blood stream. The cells introduced intoan animal are preferably cells derived from that animal, to avoid anadverse immune response. Cells also can be used that are derived from adonor animal having a similar immune background. Other cells also can beused, including those designed to avoid an adverse immunogenic response.

The target cells may be analyzed, for example for integration,transcription and/or expression of the polynucleotide or gene(s) ofinterest, the number of copies of the gene integrated, and the locationof the integration. Such analysis may be carried out at any time and maybe carried out by any methods known in the art.

Subjects in which a recombinant virus or virus-infected DCs areadministered can be analyzed for location of infected cells, expressionof the virus-delivered polynucleotide or gene of interest, stimulationof an immune response, and monitored for symptoms associated with adisease or disorder by any methods known in the art.

The methods of infecting cells disclosed above do not depend uponindividual-specific characteristics of the cells. As a result, they arereadily extended to all mammals. In some embodiments the recombinantvirus is delivered to a human or to human dendritic cells. In otherembodiments, the recombinant virus is delivered to a mouse or to mousedendritic cells. In still other embodiments, the recombinant virus isdelivered to an animal other than a human or a mouse, or to dendriticcells from an animal other than a human or a mouse.

As discussed above, the recombinant virus can be pseudotyped to conferupon it a broad host range as well as target cell specificity. One ofskill in the art would also be aware of appropriate internal promotersto achieve the desired expression of a polynucleotide or gene ofinterest in a particular animal species. Thus, one of skill in the artwill be able to modify the method of infecting dendritic cells derivedfrom any species.

The recombinant virus can be evaluated to determine the specificity ofthe targeting molecule incorporated into the virus that targetsdendritic cells. For example, a mixed population of bone marrow cellscan be obtained from a subject and cultured in vitro. The recombinantvirus can be administered to the mixed population of bone marrow cells,and expression of a reporter gene incorporated into the virus can beassayed in the cultured cells. In some embodiments, at least about 50%,more preferably at least about 60%, 70%, 80% or 90%, still morepreferably at least about 95% of transduced cells in the mixed cellpopulation are dendritic cells that express DC-SIGN.

Therapy

The methods of the present invention can be used to prevent or treat awide variety of diseases or disorders, particularly those for whichactivation of an immune response in a patient would be beneficial. Manysuch diseases are well known in the art. For example, diseases ordisorders that are amenable to treatment or prevention by the methods ofthe present invention include, without limitation, cancers, autoimmunediseases, and infections, including viral, bacterial, fungal andparasitic infections. In embodiments of the invention, a disease istreated by using recombinant viruses to deliver a gene of interest todendritic cells, wherein expression of the gene produces adisease-specific antigen and leads to stimulation of antigen-specificcellular immune responses and humoral immune responses.

In embodiments of the invention, a recombinant virus is used to deliverpolynucleotides encoding an antigen against which an immune response isdesired to dendritic cells. In some embodiments, the delivery can beachieved by contacting dendritic cells with the recombinant virus invitro, whereupon the transduced dendritic cells are provided to apatient. In some embodiments, the delivery can be achieved by deliveringthe virus to a subject for contact with dendritic cells in vivo. Thedendritic cells then stimulate antigen-specific T cells or B cells in apatient to induce cellular and humoral immune responses to the expressedantigen. In such embodiments, a patient that is suffering from a diseaseor disorder is treated by generating immune cells with a desiredspecificity.

Any antigen that is associated with a disease or disorder can bedelivered to dendritic cells using the recombinant viruses as describedherein. An antigen that is associated with the disease or disorder isidentified for preparation of a recombinant virus that targets dendriticcells. Antigens associated with many diseases and disorders are wellknown in the art. An antigen may be previously known to be associatedwith the disease or disorder, or may be identified by any method knownin the art. For example, an antigen to a type of cancer from which apatient is suffering may be known, such as a tumor associated antigen.In one aspect, the invention provides a method to deliver genes encodingtumor antigens and other necessary proteins to DCs in vivo usingengineered recombinant lentivirus. In other embodiments, an antigenrelated to the disease or disorder is identified from the patient to betreated. For example, an antigen associated with a tumor may beidentified from the tumor itself by any method known in the art. Tumorassociated antigens are not limited in any way and include, for example,antigens that are identified on cancerous cells from the patient to betreated.

Tumor associated antigens are known for a variety of cancers including,for example, prostate cancer and breast cancer. In some breast cancers,for example, the Her-2 receptor is overexpressed on the surface ofcancerous cells. Exemplary tumor antigens include, but are not limitedto: MAGE, BAGE, RAGE, and NY-ESO, which are nonmutated antigensexpressed in the immune-privileged areas of the testes and in a varietyof tumor cells; lineage-specific tumor antigens such as themelanocyte-melanoma lineage antigens MART-1/Melan-A, gp100, gp75, mda-7,tyrosinase and tyrosinase-related protein, or the prostate specificmembrane antigen (PSMA) and prostate-specific antigen (PSA), which areantigens expressed in normal and neoplastic cells derived from the sametissue; epitope proteins/peptides derived from genes mutated in tumorcells or genes transcribed at different levels in tumor compared tonormal cells, such as mutated ras, bcr/ab1 rearrangement, Her2/neu,mutated or wild-type p53, cytochrome P450 1B1, and abnormally expressedintron sequences such as N-acetylglucosaminyltransferase-V; clonalrearrangements of immunoglobulin genes generating unique idiotypes inmyeloma and B-cell lymphomas; epitope proteins/peptides derived fromoncoviral processes, such as human papilloma virus proteins E6 and E7;nonmutated oncofetal proteins with a tumor-selective expression, such ascarcinoembryonic antigen and alpha-fetoprotein. A number of tumorassociated antigens have been reviewed (see, for example,“Tumor-Antigens Recognized By T-Lymphocytes,” Boon T, Cerottini J C,Vandeneynde B, Vanderbruggen P, Vanpel A, Annual Review Of Immunology12: 337-365, 1994; “A listing of human tumor antigens recognized by Tcells,” Renkvist N, Castelli C, Robbins P F, Parmiani G. CancerImmunology Immunotherapy 50: (1) 3-15 Mar. 2001, each of which isincorporated herein by reference in its entirety.)

The antigen can also be an antigen associated with an infectiousdisease, such as, for example, HIV/AIDS. The antigen can be, forexample, gpl 20 (Klimstra, W. B., et al. 2003. J Virol 77:12022-12032;Bernard, K. A., et al. 2000. Virology 276:93-103; Byrnes, A. P., et al.1998. J Virol 72: 7349-7356, each of which is incorporated herein byreference in its entirety). Other exemplary antigens include, but arenot limited to: gag, pol, env, tat, nef and rev (Lieberman, J. et al.1997. AIDS Res Hum Retroviruses 13(5): 383-392; Menendez-Arias, L. etal. 1998. Viral Immunol 11(4): 167-181, each of which is incorporatedherein by reference in its entirety).

Examples of viral antigens include, but are not limited to, adenoviruspolypeptides, alphavirus polypeptides, calicivirus polypeptides, e.g., acalicivirus capsid antigen, coronavirus polypeptides, distemper viruspolypeptides, Ebola virus polypeptides, enterovirus polypeptides,flavivirus polypeptides, hepatitis virus (AE) polypeptides, e.g., ahepatitis B core or surface antigen, herpesvirus polypeptides, e.g., aherpes simplex virus or varicella zoster virus glycoprotein,immunodeficiency virus polypeptides, e.g., the human immunodeficiencyvirus envelope or protease, infectious peritonitis virus polypeptides,influenza virus polypeptides, e.g., an influenza A hemagglutinin,neuraminidase, or nucleoprotein, leukemia virus polypeptides, Marburgvirus polypeptides, orthomyxovirus polypeptides, papilloma viruspolypeptides, parainfluenza virus polypeptides, e.g., thehemagglutinin/neuraminidase, paramyxovirus polypeptides, parvoviruspolypeptides, pestivirus polypeptides, picorna virus polypeptides, e.g.,a poliovirus capsid polypeptide, pox virus polypeptides, e.g., avaccinia virus polypeptide, rabies virus polypeptides, e.g., a rabiesvirus glycoprotein G, reovirus polypeptides, retrovirus polypeptides,and rotavirus polypeptides.

Examples of bacterial antigens include, but are not limited to,Actinomyces polypeptides, Bacillus polypeptides, Bacteroidespolypeptides, Bordetella polypeptides, Bartonella polypeptides, Borreliapolypeptides, e.g., B. burgdorferi OspA, Brucella polypeptides,Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydiapolypeptides, Clostridium polypeptides, Corynebacterium polypeptides,Coxiella polypeptides, Dermatophilus polypeptides, Enterococcuspolypeptides, Ehrlichia polypeptides, Escherichia polypeptides,Francisella polypeptides, Fusobacterium polypeptides, Haemobartonellapolypeptides, Haemophilus polypeptides, e.g., H. influenzae type b outermembrane protein, Helicobacter polypeptides, Klebsiella polypeptides,L-form bacteria polypeptides, Leptospira polypeptides, Listeriapolypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides,Neisseria polypeptides, Neorickettsia polypeptides, Nocardiapolypeptides, Pasteurella polypeptides, Peptococcus polypeptides,Peptostreptococcus polypeptides, Pneumococcus polypeptides, Proteuspolypeptides, Pseudomonas polypeptides, Rickettsia polypeptides,Rochalimaea polypeptides, Salmonella polypeptides, Shigellapolypeptides, Staphylococcus polypeptides, Streptococcus polypeptides,e.g., S. pyogenes M proteins, Treponema polypeptides, and Yersiniapolypeptides, e.g., Y. pestis Fl and V antigens.

Examples of fungal antigens include, but are not limited to, Absidiapolypeptides, Acremonium polypeptides, Alternaria polypeptides,Aspergillus polypeptides, Basidiobolus polypeptides, Bipolarispolypeptides, Blastomyces polypeptides, Candida polypeptides,Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcuspolypeptides, Curvalaria polypeptides, Epidermophyton polypeptides,Exophiala polypeptides, Geotrichum polypeptides, Histoplasmapolypeptides, Madurella polypeptides, Malassezia polypeptides,Microsporum polypeptides, Moniliella polypeptides, Mortierellapolypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicilliumpolypeptides, Phialemonium polypeptides, Phialophora polypeptides,Prototheca polypeptides, Pseudallescheria polypeptides,Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidiumpolypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides,Sporothrix polypeptides, Stemphylium polypeptides, Trichophytonpolypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

Examples of protozoan parasite antigens include, but are not limited to,Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides,Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoonpolypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondiapolypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmaniapolypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosemapolypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides,e.g., P. falciparum circumsporozoite (PfCSP), sporozoite surface protein2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSAI c=term),and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystispolypeptides, Schistosoma polypeptides, Theileria polypeptides,Toxoplasma polypeptides, and Trypanosoma polypeptides.

Examples of helminth parasite antigens include, but are not limited to,Acanthocheilonema polypeptides, Aelurostrongylus polypeptides,Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascarispolypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillariapolypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosomapolypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides,Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydiumpolypeptides, Dirofilaria polypeptides, Dracunculus polypeptides,Enterobius polypeptides, Filaroides polypeptides, Haemonchuspolypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonellapolypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necatorpolypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides,Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagiapolypeptides, Parafilaria polypeptides, Paragonimus polypeptides,Parascaris polypeptides, Physaloptera polypeptides, Protostrongyluspolypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometrapolypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides,Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides,Toxocara polypeptides, Trichinella polypeptides, Trichostrongyluspolypeptides, Trichuris polypeptides, Uncinaria polypeptides, andWuchereria polypeptides.

Examples of ectoparasite antigens include, but are not limited to,polypeptides (including protective antigens as well as allergens) fromfleas; ticks, including hard ticks and soft ticks; flies, such asmidges, mosquitos, sand flies, black flies, horse flies, horn flies,deer flies, tsetse flies, stable flies, myiasis-causing flies and bitinggnats; ants; spiders, lice; mites; and true bugs, such as bed bugs andkissing bugs.

Once an antigen has been identified and/or selected, a polynucleotidethat encodes the desired antigen is identified. Preferably thepolynucleotide comprises a cDNA. The polynucleotides encoding theantigen are preferably introduced into target dendritic cells using arecombinant virus, more preferably a recombinant lentivirus orgammaretrovirus, including a targeting molecule that binds DC-SIGN asdescribed above. The recombinant virus first binds to the dendritic cellmembrane by way of the DC-SIGN targeting molecule, and the viral corecontaining a polynucleotide encoding the antigen subsequently enters thecytosol. The polynucleotide (e.g., one encoding the antigen) is thenpreferably integrated into the cell's genome and expressed. If contactedex vivo, the target dendritic cells are then transferred back to thepatient, for example by injection, where they interact with immune cellsthat are capable of generating an immune response against the desiredantigen. In preferred embodiments, the recombinant virus is injectedinto the patient where it transduces the targeted dendritic cells insitu. The dendritic cells then express the particular antigen associatedwith a disease or disorder to be treated, and the patient is able tomount an effective immune response against the disease or disorder.

In some embodiments, the recombinant virus contains a polynucleotidesequence encoding more than one antigen, and upon transduction of atarget dendritic cell, generates immune responses to the multitude ofantigens delivered to the cell. In some embodiments, the antigens arerelated to a single disease or disorder. In other embodiments, theantigens are related to multiple diseases or disorders.

In embodiments of the invention, DC maturation factors that activateand/or stimulate maturation of the DCs are delivered in conjunction withthe recombinant virus carrying the polynucleotide or gene of interest.In some embodiments, the DCs are activated by delivery of DC maturationfactors prior to delivery of the virus. In some embodiments, the DCs areactivated by delivery of DC maturation factors after delivery of thevirus. In some embodiments, the DCs are activated by delivery of DCmaturation factors simultaneously with delivery of the virus. In someembodiments, DC maturation factors are provided together withadministration of the virus. In other embodiments, DC maturation factorsare provided separately from administration of the virus.

In certain embodiments, one or more DC maturation factors can be encodedby one or more genes that are contained in the virus and expressed afterthe virus transducer a dendritic cell. In some embodiments, the one ormore genes encoding DC maturation factors can be included in a viralvector encoding an antigen. In further embodiments, the one or moregenes encoding DC maturation factors can be included in a viral vectorthat encodes more than one antigen. In some embodiments, the one or moregenes encoding DC maturation factors can be provided in a separatevector that is co-transfected with the viral vector encoding one or moreantigens in a packaging cell line.

In some embodiments, the methods of the present invention can be usedfor adoptive immunotherapy in a patient. As described above, an antigenagainst which an immune response is desired is identified. Apolynucleotide encoding the desired antigen is obtained and packagedinto a recombinant virus. Target dendritic cells are obtained from thepatient and transduced with a recombinant virus containing apolynucleotide that encodes the desired antigen. The dendritic cells arethen transferred back into the patient.

Vaccination

As discussed above, various engineered targeting molecules that bind theDC-SIGN surface dendritic cell marker are contemplated for use inproducing recombinant virus that delivers a gene encoding an antigen toDCs. The virus can be used to transduce DCs in vitro or in vivo forprevention of a disease or disorder. For example, the Sindbis virusenvelope glycoprotein can be engineered to bind preferentially toDC-SIGN and used to pseudotype a recombinant virus. A gene encoding anantigen against which an immune response is desired, such as for cancer(for example, Mart-1), or another disease/disorder (such as viralinfection) may be delivered to DCs using the methods described herein.In some embodiments, multiple genes encoding multiple antigens can bedelivered to DCs using the methods described herein, through the use ofmultiple viral vectors, or, preferably, a multicistronic vector system.The one or more genes for the one or more antigens may be accompanied bygenes encoding stimulatory molecules (such as GM-CSF, IL-2, IL-4, IL-6,IL-7, IL-15, IL-21, IL-23, TNFa, B7.1, B7.2, 4-1BB, CD40 ligand (CD4OL),drug-inducible CD40 (iCD40), and the like) and/or a reporter molecule(such as GFP, luciferase and the like) using multiple vectors or,preferably, a multicistronic vector system.

In some embodiments of the invention, human DCs are generated byobtaining CD34a+ human hematopoietic progenitors and using an in vitroculture method as described elsewhere (e.g., Banchereau et al. Cell 106,271-274 (2001)). Viruses bearing a targeting molecule that binds DC-SIGNare generated comprising a gene encoding an antigen against which animmune response is desired and are used to transduce human DCs.Transduction specificity and efficiency may be quantified by FACS.Maturation of DCs can be characterized by FACS analysis of up-regulationof surface marker such as MHC II.

In other embodiments, virus may be injected in vivo, where it contactsnatural DCs and delivers a polynucleotide of interest, typically a geneencoding an antigen. The amount of viral particles is at least 50×10⁶TU, and can be at least 1×10⁷ TU, at least 2×10⁷ TU, at least 3×10⁷, atleast 4×10⁷ TU, or at least 5×10⁷ TU. At selected intervals, DCs fromthe recipient's lymphoid organs may be used to measure expression, forexample, by observing marker expression, such as GFP or luciferase. Tcells from lymph nodes and spleens of virus-treated recipients may bemeasured from the magnitude and durability of response to antigenstimulation. Tissue cells other than DCs, such as epithelial cells andlymphoid cells, may be analyzed for the specificity of in vivo genedelivery.

It is widely agreed that the most effective potential method to end theAIDS epidemic (and other viral diseases) is a vaccine. To date, novaccination method against HIV has successfully passed a phase IIItrial. Thus, there is an urgent need for novel and effective vaccinationstrategies. In some embodiments of the invention, DC vaccination isused. A gene is cloned encoding a viral protein, such as those describedabove, into a viral vector. Patients are infected with virusescomprising a targeting molecule that binds DC-SIGN in DCs, preferablywith specificity such that undesired side effects are avoided. Thetargeting molecule can be, for example, an engineered Sindbis virusenvelope glycoprotein, and the administration of virus can be carriedout, for example, by injection. In an animal model, molecularly clonedHIV reporter viruses (NFNSZ-r-HSAS, NL-r-HSAS) and clinical isolates maybe used to challenge the animals by tail vein injection. Evidence ofinfection may be monitored over time in splenocytes, lymph nodes, andperipheral blood. PCR for HIV-gag protein and FACS for HAS in thereporter viruses may be used to test for viral integration andreplication. Productive in situ DC vaccination may increase resistanceto a HIV challenge. See Examples 17-20.

In some embodiments, dendritic cells transduced with a recombinant virusas described herein are provided for the prevention of or treatment of adisease or disorder. In preferred embodiments, the dendritic cellsexpress an antigen against which an immune response is desired. Theantigen is typically one that is not normally expressed in a dendriticcell but is expressed after the target cell is transduced with therecombinant virus containing a polynucleotide encoding the antigen. Insome embodiments, the dendritic cells further express a DC maturationfactor which is provided to the dendritic cell by a recombinant virus asdescribed herein.

In some aspects of the invention, an adjuvant is administered inconjunction with a recombinant virus of the invention. The adjuvant maybe administered with the recombinant virus, before the recombinantvirus, or after the recombinant virus.

A variety of adjuvants can be used in combination with the recombinantvirus of the invention to elicit an immune response to the antigenencoded to the recombinant virus. Preferred adjuvants augment theintrinsic response to an antigen without causing conformational changesin the antigen that affect the qualitative form of the response.Preferred adjuvants include alum, 3 De-O-acylated monophosphoryl lipid A(MPL) (see GB 2220211). QS21 is a triterpene glycoside or saponinisolated from the bark of the Quillaja Saponaria Molina tree found inSouth America (see Kensil et al., in Vaccine Design: The Subunit andAdjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S.Pat. No. 5,057,540). Other adjuvants are oil in water emulsions (such assqualene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engi.J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today,Nov. 15, 1998). Alternatively, AP can be coupled to an adjuvant. Forexample, a lipopeptide version of AP can be prepared by couplingpalmitic acid or other lipids directly to the N-terminus of AP asdescribed for hepatitis B antigen vaccination (Livingston, J. Immunol.159, 1383-1392 (1997)). However, such coupling should not substantiallychange the conformation of AP so as to affect the nature of the immuneresponse thereto. Adjuvants can be administered as a component of atherapeutic composition with an active agent or can be administeredseparately, before, concurrently with, or after administration of thetherapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvantscan be used with or without other specific immunostimulating agents suchas MPL or 3-DMP, QS21, polymeric or monomeric amino acids such aspolyglutamic acid or polylysine. Another class of adjuvants isoil-in-water emulsion formulations. Such adjuvants can be used with orwithout other specific immunostimulating agents such as muramyl peptides(e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alaninel-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalane, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another classof preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS21,Aquila, Worcester, Mass.) or particles generated therefrom such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA). Other adjuvants include cytokines, such as interleukins(IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF),tumor necrosis factor (TNF).

An adjuvant can be administered with the recombinant virus of theinvention as a single composition, or can be administered before,concurrent with or after administration of the recombinant virus of theinvention. Immunogen and adjuvant can be packaged and supplied in thesame vial or can be packaged in separate vials and mixed before use.Immunogen and adjuvant are typically packaged with a label indicatingthe intended therapeutic application. If immunogen and adjuvant arepackaged separately, the packaging typically includes instructions formixing before use. The choice of an adjuvant and/or carrier depends onthe stability of the vaccine containing the adjuvant, the route ofadministration, the dosing schedule, the efficacy of the adjuvant forthe species being vaccinated, and, in humans, a pharmaceuticallyacceptable adjuvant is one that has been approved or is approvable forhuman administration by pertinent regulatory bodies. For example,Complete Freund's adjuvant is not suitable for human administration.Alum, MPL and QS21 are preferred. Optionally, two or more differentadjuvants can be used simultaneously. Preferred combinations includealum with MPL, alum with QS21, MPL with QS21, and alum, QS21 and MPLtogether. Also, Incomplete Freund's ajuvant can be used (Chang et al.,Advanced Drug Delivery Reviews 32, 173-186 (1998)), optionally incombination with any of alum, QS21, and MPL and all combinationsthereof.

Pharmaceutical Compositions and Kits

Also contemplated herein are pharmaceutical compositions and kitscontaining a recombinant virus provided herein and one or morecomponents. Pharmaceutical compositions can include a recombinant virusprovided herein and a pharmaceutical carrier. Kits can include thepharmaceutical compositions and/or combinations provided herein, and oneor more components, such as instructions for use, a device foradministering a compound to a subject, and a device for administering acompound to a subject.

1. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions containing a virusprovided herein and a suitable pharmaceutical carrier. Pharmaceuticalcompositions provided herein can be in various forms, e.g., in solid,liquid, powder, aqueous, or lyophilized form. Examples of suitablepharmaceutical carriers are known in the art. Such carriers and/oradditives can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Stabilizing agents suchas lipids, nuclease inhibitors, polymers, and chelating agents canpreserve the compositions from degradation within the body.

2. Kits

The recombinant viruses provided herein can be packaged as kits. Kitscan optionally include one or more components such as instructions foruse, devices, and additional reagents, and components, such as tubes,containers and syringes for practice of the methods. Exemplary kits caninclude the viruses provided herein, and can optionally includeinstructions for use, a device for detecting a virus in a subject, adevice for administering the virus to a subject, and a device foradministering a compound to a subject.

Kits comprising polynucleotides encoding a gene of interest (typicallyan antigen) are also contemplated herein. In some embodiments, the kitincludes at least one plasmid encoding virus packaging components andvector encoding a targeting molecule that is engineered to binddendritic cells, preferably with specificity. In some embodiments, thekit includes at least one plasmid encoding virus packaging components, avector encoding a targeting molecule that is engineered to binddendritic cells and a vector encoding at least one DC maturation factor.

Kits comprising a viral vector encoding a gene of interest (typically anantigen) and optionally, a polynucleotide sequence encoding a DCmaturation factor are also contemplated herein. In some embodiments, thekit includes at least one plasmid encoding virus packaging componentsand vector encoding a targeting molecule that is engineered to binddendritic cells.

In one example, a kit can contain instructions. Instructions typicallyinclude a tangible expression describing the virus and, optionally,other components included in the kit, and methods for administration,including methods for determining the proper state of the subject, theproper dosage amount, and the proper administration method, foradministering the virus. Instructions can also include guidance formonitoring the subject over the duration of the treatment time.

Kits provided herein also can include a device for administering a virusto a subject. Any of a variety of devices known in the art foradministering medications or vaccines can be included in the kitsprovided herein. Exemplary devices include, but are not limited to, ahypodermic needle, an intravenous needle, a catheter, a needle-lessinjection device, an inhaler, and a liquid dispenser, such as aneyedropper. Typically, the device for administering a virus of the kitwill be compatible with the virus of the kit; for example, a needle-lessinjection device such as a high pressure injection device can beincluded in kits with viruses not damaged by high pressure injection,but is typically not included in kits with viruses damaged by highpressure injection.

Kits provided herein also can include a device for administering acompound, such as a DC activator or stimulator, to a subject. Any of avariety of devices known in the art for administering medications to asubject can be included in the kits provided herein. Exemplary devicesinclude a hypodermic needle, an intravenous needle, a catheter, aneedle-less injection, but are not limited to, a hypodermic needle, anintravenous needle, a catheter, a needle-less injection device, aninhaler, and a liquid dispenser such as an eyedropper. Typically thedevice for administering the compound of the kit will be compatible withthe desired method of administration of the compound.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

Example 1 Engineering of a DC-Specific Targeting Molecule

Lentiviral vectors can be rationally engineered to make them capable oftransducing DCs in a cell-specific manner. Certain subsets of DCs bearon their surface the DC-SIGN protein (Geijtenbeek, T. B., et al. 2000;Geijtenbeek, T. B., et al. 2000, supra), a C-type lectin-like receptorcapable of rapid binding and endocytosis of materials (Geijtenbeek, T.B., et al. 2004, supra.), which can be used as a targeting receptor onDCs. Sindbis virus (SV)—a member of the Alphavirus genus and theTogaviridae family—is able to infect DCs through DC-SIGN (Klimstra, W.B., et al. 2003. J. Virol. 77: 12022-12032, which is incorporated hereinby reference in its entirety). However, the canonical viral receptor forthe laboratory strain of SV is cell-surface heparan sulfate (HS), whichis expressed by many cell types (Strauss, J. H., et al. 1994. Arch.Virol. 9: 473-484; Byrnes, A. P., and D. E. Griffin. 1998. J. Virol. 72:7349-7356, each of which is incorporated herein by reference in itsentirety). Taking advantage of the physical separation of the tworeceptor-binding sites on the SV envelope glycoprotein (hereafterdesignated as SVG), the receptor was engineered to be blind to itscanonical binding target HS and to leave intact its ability to interactwith DC-SIGN (FIG. 1). Once it is incorporated onto a viral surface,this mutant glycoprotein is able to mediate infection of DCs but notother cells.

The cDNA for wild-type SVG was obtained from the laboratory of Dr. J. H.Strauss laboratory at the California Institute of Technology and clonedinto the pcDNA3 vector (Invitrogen) by PCR to generate plasmid pSVG. Aten-residue tag (MYPYDVPDYA) sequence was inserted into E2 proteinbetween amino acids 71 and 74 by PCR mutagenesis to disrupt the HSbinding site (Karavans, G., et al. 1998. Crit Rev Oncol Hemat 28: 7-30;Lavillete, D., et al. 2001. Curr Opin Biotech 12: 461-466; Russell, S.J. and F. L. Cosset. 1999. J Gene Med 1: 300-311; Sandrin, V., et al.2003. Curr Top Microbiol 281: 137-178; Verhoeyen, E. and F. L. Cosset.2004. J Gene Med 6: S83-S94, each of which is incorporated herein byreference in its entirety). An available antibody against the insertedtag sequence provided the ability to monitor the expression of themodified SVG. In order to further decrease the HS-specific infection,several critical residues were identified as being involved in bindingto HS molecules (Coffin, J. M., et al. 1997. Retroviruses. New York:Cold Spring Harbor Laboratory Press; Battini, J. L., et al. 1998. JVirol 72:428-435; Valsesiawittmann, S., et al. 1994. J Virol68:4609-4619; Wu, B. W., et al. 2000. Virology 269: 7-17; Cosset, F. L.,et al. 1995. J Virol 69:6314-6322; Kayman, S. C., et al. 1999. J Virol73: 1802-1808; Lorimar, I. A. J. and S. J. Lavictoire. 2000. J ImmunolMethods 237:147-157; Barnett, A. L., et al. 2001. Proc Nat Acad Sci USA98: 4113-4118; Benedict, C. A., et al. 2002. Hum Gene Ther 10:545-557;Gollan, T. J. and M. R. Green. 2002. J Virol 76:3558-3563, each of whichis incorporated herein by reference in its entirety). Two such residueswere mutated into alanines (157KE158 to 157AA158).

An additional deletion was introduced to the E3 glycoprotein of SVG toremove amino acids 61-64. This modified SVG was designated as SVGmu (SEQID NO: 11). The cDNA for SVGmu was cloned downstream of the CMV promoterin the pcDNA3 vector (designated as pSVGmu, SEQ ID NO: 3).

Example 2 Preparation of Recombinant Virus Containing the DC-SpecificTargeting Molecule

Preparation of the recombinant SVGmu-pseudotyped lentivirus wasconducted by standard calcium phosphate-mediated transient transfectionof 293T cells with the lentiviral vector FUGW (SEQ ID NO:1) or itsderivatives, the packaging constructs encoding gag, pol and rev genes,and pSVGmu (Example 1). FUGW is a self-inactivating lentiviral vectorcarrying the human ubiquitin-C promoter to drive the expression of a GFPreporter gene (Lois, C., et al. 2002. Science 295: 868-872, which isincorporated herein by reference in its entirety). The lentiviraltransfer vectors (FUGW and its derivatives) used in these studies arethird generation HIV-based lentiviral vectors, in which most of the U3region of the 3′ LTR is deleted, resulting in a self-inactivating 3′-LTR(SIN).

For the transient transfection of 293T cells, 293T cells cultured in6-cm tissue culture dishes (Coming or BD Biosciences) were transfectedwith the appropriate lentiviral transfer vector plasmid (5 jig), alongwith 2.5 [ig each of the envelope plasmid (SVG, SVGmu, Eco, or VSVG) andthe packaging plasmids (pMDLg/pRRE and pRSV-Rev). The viral supernatantswere harvested 48 and 72 hours post-transfection and filtered through a0.45-pm filter (Coming). To prepare concentrated viral vectors for invivo study, the viral supernatants were ultracentrifugated (Optima L-80K preparative ultracentrifuge, Beckman Coulter) at 50,000×g for 90 min.The pellets were then resuspended in an appropriate volume of cold PBS.

The resultant viruses pseudotyped with SVGmu are hereafter referred toas FUGW/SVGmu. Control viruses enveloped with the wild-type SVGglycoprotein are hereafter referred to as FUGW/SVG.

Example 3 Confocal Imaging of Packaged Recombinant Virus

GFP-vpr-labeled lentivectors were produced as described in Example 2,except with use of FUW lentivector (which does not contain the GFPreporter gene) and with a separate plasmid encoding GFP-vpr (2.5 lug).Fresh viral supernatant was overlaid on polylysine-coated coverslips ina 6-well culture dish and centrifuged at 3,700×g at 4° C. for 2 hoursusing a Sorvall Legend RT centrifuge. The coverslips were rinsed withcold PBS twice and immunostained by anti-HA-biotin antibody (MiltenyiBiotec) and Cy5-streptavidin (Invitrogen). Fluorescent images were takenby a Zeiss LSM 510 laser scanning confocal microscope equipped withfilter sets for fluorescein and Cy5. A plan-apochromat oil immersionobjective (63×/1.4) was used for imaging.

FIG. 2 shows the results of the confocal imaging of the recombinantvirus produced by the protocol. (The scale bar represents 2 um.)Particles in the “GFP” slide are stained green, particles in the “SVGmu”slide are stained red, and particles in the “Merged” slide are stainedgreen where only GFP is expressed, red where only SVGmu is expressed,and yellow/yellow-orange where GFP and SVGmu are both expressed. Over90% of the GFP-labeled particles contained SVGmu. Thus, the productionof lentiviral particles displaying SVGmu was confirmed through confocalimaging.

Example 4 Preparation of DC-Sign Cell Lines

To facilitate the study of targeted transduction, DC-SIGN cell linesexpressing human DC-SIGN (hereafter referred to as 293T.hDCSIGN) andmurine DC-SIGN (hereafter referred to as 293T.mDCSIGN) were constructed.The 293T.hDCSIGN and 293T.mDCSIGN cell lines were generated by stabletransduction of parental 293T cells with a VSVG-pseudotyped lentivector.The cDNAs for human DC-SIGN and murine DC-SIGN were amplified fromplasmids pUNO-hDCSIGN1Aa and pUNO-mDCSIGN (InvivoGene) and cloneddownstream of the human ubiquitin-C promoter in the lentiviral plasmidFUW to generate FUW-hDCSIGN (SEQ ID NO: 5) and FUW-mDCSIGN (SEQ ID NO:6), respectively. The lentivectors were then pseudotyped with VSVG andused to transduce 293T cells. The resulting cells were subjected toantibody staining (anti-human DC-SIGN antibody from BD Biosciences andanti-murine DC-SIGN from eBioscience) and cell sorting to yield auniform population of DC-SIGN⁺ 293T.hDCSIGN and mDC-SIGN⁺ 293T.mDCSIGNcell lines

Flow cytometry showed that DC-SIGN was expressed in virtually all of the293T.hDCSIGN and 293T.mDCSIGN cells of the cell lines (FIG. 3A). In eachdiagram, the solid lines (unfilled area) represents expression ofDC-SIGN in the 293T DC-SIGN cell lines, and the shaded area representsthe background staining of non-transduced 293T cells.

Example 5 Evaluation of the DC-Sign Specific Recombinant Virus byTransduction of DC-Sign Cell Lines

To assess the transduction efficiency and specificity of FUGW/SVG orFUGW/SVGmu (Example 2), the viruses were used to transduce the293T.hDCSIGN and 293T.mDCSIGN cell lines (Example 4). Transductionefficiency was measured by GFP expression within the cell lines.

Target cells (293T.hDCSIGN, 293T.mDCSIGN, or 293T cells; 0.2×10⁶ perwell) were seeded in a 24-well culture dish (Coming or BD Biosciences)and spin-infected with viral supernatants (1 ml per well) at 2,500 rpmand 30° C. for 90 min by using a Sorvall Legend centrifuge.Subsequently, the supernatants were replaced with fresh culture mediumand incubated for 3 days at 37° C. with 5% of CO₂. The percentage ofGFP⁺ cells was measured by flow cytometry. The transduction titer wasdetermined by the dilution ranges that exhibited a linear response

Flow cytometry showed that FUGW/SVG (containing the wild-type SVGenvelope glycoprotein) had similar transduction efficiency (11-16%transduction) towards the three target cell lines (293T, 293T.hDCSIGN,and 293T.mDCSIGN) (FIG. 3B). This indicates that that SVG has broadspecificity and the presence of DC-SIGN on the cell surface does notmarkedly alter the transduction ability of a SVG-pseudotyped lentiviralvector. In contrast, the FUGW/SVGmu vector (containing the mutant SVGenvelope glycoprotein) could specifically transduce 293T.hDCSIGN and293T.mDCSIGN cells with a 42% and 34% transduction efficiency,respectively, but not the 293T cells (FIG. 3B). These resultsdemonstrate that a pseudotyped lentiviral vector displaying SVGmu canspecifically transduce cells expressing either human or murine DC-SIGN.Furthermore, the mutant SVG gave more efficient transduction ofDC-SIGN-expressing cells than of wild type SVG.

The stable integration of the FUGW lentiviral vector in the transducedcells was confirmed by PCR analysis of the genomic integration of theGFP reporter gene. To demonstrate that the specific transduction wasmediated by DC-SIGN, the addition of soluble anti-human DC-SIGN antibodyto the FUGW/SVGmu viral supernatant before its exposure to 293T.hDCSIGNcells reduced the transduction efficiency (data not shown). The specifictiter of FUGW/SVGmu for 293T.mDCSIGN was estimated to be 1×10⁶ TU(Transduction Units)/ml. The titer of FUGW/SVGmu for 293T.hDCSIGN wasestimated to be 1-2×10⁶ TU/ml.

Example 6 Evaluation of the Recombinant Virus In Vitro

To investigate the specificity of the engineered lentivector fortransduction of dendritic cells (DCs) expressing DC-SIGN, total bonemarrow (BM) cells were isolated from mice and transduced directly withthe FUGW/SVGmu viral vector (Example 2). A protocol to generate mouseDCs from progenitors grown in BM cultures was adapted for use in theexperiment (Buchholz, C. J., et al. 1998. Nat Biotech 16:951-954, whichis incorporated herein by reference in its entirety).

Total bone marrow cells were harvested from B6 female mouse (CharlesRiver Breeding Laboratories), and BMDCs were generated as describedelsewhere (Yang, L. and D. Baltimore. 2005. Proc. Natl. Acad. Sci. USA102: 4518-4523, which is incorporated herein by reference in itsentirety). Either total BM cells or BMDCs were plated in a 24-wellculture dish (2×10⁶ cells per well), and spin-infected with viralsupernatant (1 ml per well) at 2,500 rpm and 30° C. for 90 min using aSorvall RT7 centrifuge. After the spin, the supernatant was removed andreplaced with fresh RPMI medium containing 10% FBS and GM-CSF (1:20J558L conditioned medium). The cells were cultured for 3 days and wereanalyzed for GFP expression using flow cytometry.

The BM cells isolated from mice were transduced directly with eitherFUGW/SVGmu viral vector or with a control vector. For the control, anecotropic murine leukemia virus glycoprotein (Eco)-enveloped lentivector(FUGW/Eco) was used; vector enveloped with Eco can infect rodent cellswith a broad specificity. Three days post-infection, the transductionefficiency was analyzed by flow cytometry (FIG. 4A). Approximately 9% ofthe cells in the mixed BM cultures were DCs (as indicated by theexpression of CD11c), of which most (approximately 80%) were DC-SIGNhigh (data not shown). It was observed that 12% of the total BM cellswere GFP positive (GFP⁺) upon FUGW/SVGmu transduction (FIG. 4A). Whengated on GFP⁺ cells, it was observed that up to 95% of the transducedcells were DC-SIGN and CD11c double-positive (DC-SIGN⁺CD11c⁺),indicating that FUGW/SVGmu specifically transduces DCs expressingDC-SIGN and not other cell types in the bone marrow. In contrast,although 68% of total BM cells were GFP-positive after exposure toFUGW/Eco, only 9% of the transduced cells were DCs, within which 6.5%were DC-SIGN⁺.

The stable transduction of FUGW/SVGmu was verified by Alu PCR analysis(Butler, S. L., et al. 2001. Nat. Med. 7: 631-634, which is incorporatedherein by reference in its entirety) of the genomic integration of theLTR of the lentivector backbone. In addition, we used FUGW/SVGmu totransduce primary T and B cells harvested from mouse spleen andvirtually no transduction was detected (FIG. 4B), indicating remarkabletransduction specificity.

The efficiency of the lentivector bearing SVGmu to transduce invitro-cultured, bone marrow (BM)-derived DCs (BMDCs) was also tested.Bone marrow (BM)-derived DCs (BMDCs) were generated as described aboveby culturing in the presence of granulocyte-macrophagecolony-stimulating factor (GM-CSF) for 6 days. The cells were thenexposed to either the FUGW/SVGmu or FUGW/Eco lentivector. Flow cytometryof the BMDCs on day 3 post-transduction showed that FUGW/Eco transducedboth CD11c⁺ DCs (33%) and CD11c⁻ cells (7.6%) (FIG. 5), which isconsistent with the wide tropism of Eco. On the contrary, FUGW/SGVmuonly transduced CD11c⁺ DCs (32.7%), and no GFP⁺ cells were detectedamong the CD11c⁻ cells (FIG. 5), indicating that FUGW/SVGmu canspecifically modify BMDCs.

These results thus collectively demonstrate that the engineeredrecombinant lentivectors bearing SVGmu can specifically transduce DCs invitro and that the targeted transduction is correlated with theexpression of DC-SIGN on the surface of DCs.

Example 7 Effect of Recombinant Virus on Activation of Dendritic CellsIn Vitro

The recombinant lentivirus was further examined to determine whether itcould specifically target, transduce and activate DCs into mature DCs.The surface up-regulation of the co-stimulatory molecule B7.2 (CD86) andthe MHC class II molecule I-A^(b), which are considered to be signaturesof DC activation (Steinman, R. M., et al. 2003. Annu. Rev. Immunol. 21:685-711, which is incorporated herein by reference in its entirety), wasmeasured in DCs exposed to recombinant virus. BMDCs were generated andinfected with FUGW/SVGmu as described in Example 6. LPS at aconcentration of 1 μg/ml was also added overnight for further activationof transduced BMDCs.

Flow cytometry of BMDCs 3 days post-transduction showed that treatmentwith FUGW/SVGmu elevated the expression of DC activation markers, CD86and I-A^(b), on GFP positive DCs, as compared to GFP negative DCs (FIG.6, top panel). The shaded area indicates GFP negative (untransduced)cells, and the solid line (unfilled area) indicates GFP positive(transduced) cells. It was observed that the targeted transduction ofBMDCs synergized with lipopolysaccharide (LPS) treatment to furthermature DCs (FIG. 6, bottom panel). This indicates that the targetedtransduction can either work alone or in combination with other DCmaturation factors to induce DC activation.

Example 8 Targeting of Dendritic Cells In Vivo by Recombinant Virus

The proof of whether this methodology can be used for vaccination can beexamined by in vivo experimentation. To test whether engineeredlentivectors bearing SVGmu could target DCs in vivo, the recombinant andconcentrated lentivector FUGW/SVGmu (50×10⁶ TU resuspended in 200 IAPBS) was injected subcutaneously into the left flank of the C57BL/6female mice (B6, Charles River Breeding Laboratories) close to aninguinal lymph node (within 1 cm range). The left inguinal lymph nodeand the equivalent lymph node at the opposite site were isolated forsize examination on day 3 post-injection. The cells were harvested fromthese nodes and their total numbers were counted. The percentage of GFP⁺DCs was analyzed by flow cytometry on cells stained with anti-CD11cantibody (BD Biosciences).

On day 3, a significant enlargement of the left inguinal lymph nodeclose to the injection site was observed (FIG. 7A, left image), and thecell number in this lymph node increased more than 10-fold, comparedwith the equivalent lymph node at the opposite side or lymph nodes froma naïve mouse (FIG. 7B). This indicates that vector administration canenhance trafficking and proliferation of lymphocytes in a nearby lymphnode.

Flow cytometry indicated that approximately 3.8% of the total CD11c⁺cells in the left inguinal lymph node cells were GFP⁺ DCs (FIG. 7C),which appear to have migrated from the injection site. This isconsidered a remarkably large effect from one subcutaneous injection ofvector and demonstrates that the recombinant virus is effectivelyinfecting DCs in vivo.

Example 9 Evaluation of the Specificity of Recombinant Virus by In VivoTransduction

To examine the in vivo specificity of the DC-targeted lentivector, alentiviral vector encoding a firefly luciferase was constructed. ThecDNA of firefly luciferase was amplified from pGL4.2LucP (Promega) andcloned into FUGW (Lois, C. et al. 2002. supra.) to replace GFP, yieldingthe construct Flue (SEQ ID NO: 4) (FIG. 22A). The luciferase reportergene was then used to visualize the in vivo transduction of the tissuecells using standard protocols of bioluminescence imaging (BLI).

The recombinant lentivector (hereafter referred to as Fluc/SVGmu) wasinjected subcutaneously at the left flank of the mouse. In anothermouse, a lentivector pseudotyped with vesicular stomatitis viralglycoprotein (hereafter referred to as Fluc/VSVG) was injected as anon-specific vector control. Vector-treated mice were then imagednon-invasively using BLI. Fluc/VSVG-treated mice had a strong andpermanent signal at the injection site, indicating that non-specifictissues were transduced to express luciferase (FIG. 22B). This isconsistent with the fact that VSVG-enveloped virus has broadspecificity. In contrast, no significant signal was detected at theinjection site of Fluc/SVGmu-treated mice (FIG. 22B), indicating thatthe lentivector bearing SVGmu had a relatively stringent targetspecificity. At no time was luminescence signal able to be detected inthe targeted mice, likely due to the rare and sparse distribution of theDCs, which is beyond the sensitivity of the current BLI method.

After one month, the mice injected with Fluc/SVGmu were subjected tobiodistribution analysis by quantitative RT-PCR and no detectable copyof the lentivector was observed in all isolated organs (heart, liver,spleen, kidney, gonad, lung, skin, lymph node), verifying the lack ofnon-specific infection in the animals and thus the specificity of thetargeted vector for DCs.

Example 10 In Vitro Antigen Delivery by Recombinant Virus

To determine whether the targeted transduction of DCs by a recombinantlentivector could be used to effectively deliver antigen genes to DCsfor stimulation of antigen-specific CD8⁺ and CD4⁺ T cell responses, alentivector expressing the model antigen, chicken ovalbumin (OVA), wasconstructed. In C57BL/6J (B6) mice, OVA is a well-characterized targetantigen for the CD8⁺ T-cell receptor OT1, which specifically bindsOVA257-269 (designated as OVAp), and for the CD4⁺ T-cell receptor OT2,which specifically binds OVA323_339 (designated as OVAp*) (Yang, L. andD. Baltimore. 2005. Proc. Nail. Acad. Sci. USA 102: 4518-4523, which isincorporated by reference in its entirety). The lentivector expressingOVA (FOVA (SEQ ID NO:2), FIG. 8, top) was constructed from FUGW (FIG. 8,bottom) by replacing the GFP with the eDNA of chicken ovalbumin.

The BMDCs (Example 6) were transduced on day 6 of culture with eitherrecombinant lentivirus FOVA/SVGmu or control recombinant lentivirusFUGW/SVGmu (encoding a non-relevant reporter gene GFP). The day 6 BMDCswere spin-infected with viral supernatant, and cultured for anadditional 3 days. On day 9, the non-adherent cells were collected andre-cultured in RPMI medium containing 10% FBS, GM-CSF (1:20 J558Lconditional medium), and 1 pg/ml LPS (Sigma). On day 10, the cells werecollected and used for T cell stimulation. The modified BMDCs weredesignated as DC/FOVA and DC/FUGW, depending on the lentivector used fortransduction. In parallel, non-adherent cells were collected fromnon-transduced day 9 BMDC culture, and were re-cultured in the samemedium (RPMI containing 10% FBS, GM-CSF and LPS). On day 10, the cellswere collected and loaded with either OVAp (OVA257-269, specificallybound by OT1 T-cell receptors, hereafter referred to as DC/OVAp) orOVAp* (OVA₃₂₃ _(_) ₃₃₉, specifically bound by OT2 T-cell receptors,hereafter referred to as DC/OVAp*), and used as positive controls for Tcell stimulation. To examine the ability of vector-transduced BMDCs toprocess and present the transgenic OVA antigen, spleen cells werecollected from the OT1 and OT2 transgenic mice and cultured with thelentivector-transduced BMDCs, or BMDCs loaded with either OVAp or OVAp*,at the indicated ratio. Three days later, the supernatant was collectedand assayed for IFN-y production using ELISA and the cells werecollected and analyzed for their surface activation markers using flowcytometry. T cell proliferation was assayed using [³H] thymidineincorporation.

After a three-day coculture with varying ratios of DC/FOVA to transgenicT cells, OT1 T cells responded vigorously as measured by the release ofIFN-y (FIG. 10A) and T cell proliferation (FIG. 10B). As expected, noobvious OVA response was detected using DC/FUGW (FIGS. 10A and 10B). Itwas also observed that the transgenic expression of OVA was even moreefficient than peptide-loading for stimulation of an OT1 T cellresponse, which is consistent with the notion that MHC class I favorsthe presentation of endogenously produced peptides. Flow cytometryshowed that the activated OT1 T cells exhibited the typical effectorcytotoxic T cell phenotype (CD25^(±)CD69^(±)CD62L^(I°w)CD44^(h1)g^(h))after stimulation by either DC/FOVA or DC/OVAp (FIG. 9).

When the DCs were co-cultured with OT2 CD4⁺ T cells, T cell activationwas also observed, as indicated by changes in the surface markers (FIG.11) and the production of IFN-y (FIG. 12). However, stimulation of CD4⁺cells was not as pronounced as that of CD8⁺ cells, presumably due to theless efficient presentation of endogenous antigen peptides to the MHCclass II molecules. By modifying the cellular localization of OVAantigen to direct it to MHC class H presentation pathway, an enhancementof CD4 stimulation was achieved that was even better than that ofpeptide-pulsed DCs (data not shown).

These results show that the method of DC targeting through lentivectorinfection can effectively deliver antigens to DCs and stimulate bothCD8⁺ and CD4⁺ T cell responses.

Example 11 In Vivo Antigen Delivery by Recombinant Virus

To determine if DCs targeted with lentivectors could activateantigen-specific T cells in vivo, a method of T-cell receptor (TCR) genetransfer into murine hematopoietic stem cells (HSCs) was used togenerate antigen-specific and TCR-engineered T cells in mice, asdescribed elsewhere (Yang, L. and D. Baltimore, D. 2005. supra.). Atricistronic retroviral vector MIG-0T1 co-expressing OT1 TCRa and TCRi3,along with the GFP marker (FIG. 13A) was constructed.

Briefly, B6 female mice (Charles River Breedling Laboratories) weretreated with 250 p,g of 5-flurouracil (Sigma). Five days later, bonemarrow (BM) cells enriched with HSCs were harvested from the tibia andfemur and cultured in a 24-well culture plate (2×10⁶ cells per well) inBM culture medium (RPMI containing 10% FBS, 2 Ong/ml rmIL-3, 5 Ong/m1rmIL-6 and 5 Ong/ml rmSCF (PeproTech)). On day 1 and day 2 of theculture, the cells were spin-infected with the MIG-0T1 retroviral vectorpseudotyped with Eco (2 ml viral supernatant per well) at 2,500 rpm and30° C. for 90 min. After each spin, the supernatant was removed andreplaced with fresh BM culture medium. On day 3, the transduced BM cellswere collected and transferred into B6 recipient mice receiving 1,200rads of total body irradiation. Eight weeks post-transfer, the mice wereused for the in vivo immunization study. Each mouse received one dose ofsubcutaneous injection of 10×10⁶ TU of targeting lentivector. Seven dayslater, spleen and lymph node cells were harvested and analyzed for thepresence of OT1 T cells and their surface activation markers using flowcytometry.

Eight weeks post-transfer, analysis of the peripheral T cells of thereconstituted mice showed that approximately 5% of the CD8⁺ T cells wereGFP⁺OT1⁺ (FIG. 13B). Some of the reconstituted mice were immunized viasubcutaneous injection of the same dose (10×10⁶ TU) of either FOVA/SVGmu(Example 10) or FUGW/SVGmu (Example 2). Analysis of GFP⁺OT1⁺ T cellsharvested from peripheral lymphoid organs 7 days later showed that thetargeted DC immunization by FOVA/SVGmu doubled the number of OT1 T cellsas compared to the control mice, which were either not immunized orimmunized with FUGW/SVGmu (FIG. 14B). The GFP⁺OT1⁺ T cells derived fromFOVA/SVGmu-immunized mice exhibited an effector memory phenotype(CD69^(16w)CD62^(hi)g^(h)CD44^(hI)g^(h)), indicating these cells havegone through a productive immune response (FIG. 14A).

These results demonstrate that a recombinant lentivector bearing surfaceSVGmu can target DCs in vivo to efficiently stimulate antigen-specific Tcells and induce a strong immune response.

Example 12 Induction of In Vivo CTL and Antibody Responses by DirectAdministration of Recombinant Virus

Studies were conducted on the efficacy of the in vivo DC targeting forinducing an antigen-specific CD8⁺ cytotoxic T lymphocyte (CTL) responseand antibody response through the administration of the targetinglentivector to naïve, wild-type mice.

Wild-type B6 mice (Charles River Breeding Laboratories) were given asingle injection of targeting lentivector (50+10⁶ TU of FUGW/SVG orFOVA/SVGmu) subcutaneously on the right flank at the indicated dose. Onday 7 and day 14 post-immunization, blood was collected from theimmunized mice through tail bleeding, and the serum anti-OVA IgG wasmeasured using ELISA. On day 14, spleen and lymph node cells wereharvested and analyzed for the presence of OVA-specific T cells andtheir surface activation markers using flow cytometry.

The presence of OVA-specific T cells was measured by measuring cytokinesecretion and tetramer staining. At day 14 post-injection, T cellsharvested from peripheral lymphoid organs were analyzed. Lentivectortargeting to native DCs was able to elicit OVA-responsive CD8⁺ T cellsin both the lymph node (data not shown) and spleen (FIG. 23).Administration of a single dose of recombinant FOVA/SVGmu was sufficientto generate CD8⁺ T cells, which could be primed to secrete IFN-y uponOVAp restimulation (FIG. 23). Administration of the control vectorFUGW/SVGmu failed to generate any OVAp-specific responses (FIG. 23). Tofurther evaluate the magnitude of responses, the OVAp-specific CD8⁺ Tcells was measured by MHC class I tetramer staining. A high frequency ofOVAp-specific T cells (>6%) was obtained following a single doseinjection (FIG. 15); no tetramer-positive cells were detected in themice treated with FUGW/SVGmu (FIG. 15). The data generated by tetramerquantitation correlated well with the analysis of CD8⁺ effector cellsassayed by intracellular IFN-y staining (FIG. 23). Phenotype analysis ofthese OVAp-positive T cells showed that these cells displayed thesurface characteristics of effector memory T cells(CD25¹″^(/)CD69¹″^(/)CD62L^(hi)g^(h)CD44^(h)′g^(h)) (FIG. 17A).

To investigate the dose response of lentivector administration, doses ofFOVA/SVGmu ranging from 100×10⁶ TU to 3×10⁶ TU were injectedsubcutaneously and OVAp-specific T cells in the spleen were measured atday 14 post-injection. An exceptionally high frequency (12%) ofOVAp-specific CD8⁺ T cells was detected at the dose of 100×10⁶ TU (FIG.16A). The percentage of OVAp-specific cells correlated proportionatelywith the amount of recombinant vector administered (FIG. 16B). A plateauin the dose response was not achieved with the doses that were tested,indicating that further enhancement can be achieved by increasing theamount of vector injected and/or the frequency of injection.

Further, the serum IgG levels specific for OVA in mice were examined onthe 7th and 14th days after immunization with FOVA/SVGmu (50×10⁶ TU).The IgG serum titer was 1:10,000 on day 7 and 1:30,000 on day 14 (FIG.17B). This is a rather impressive antibody response for a single doseinjection without additional adjuvant or other stimuli, indicating thattargeted lentivector immunization can also elicit significant B cellsecretion of antigen-specific antibodies.

These results show that in vivo administration of a DC-targetinglentivector can induce both cellular and humoral immune responsesagainst the delivered antigen.

Example 13 Generation of Anti-Tumor Immunity Preventive Protection

The anti-tumor immunity generated after an in vivo administration ofDC-targeted lentivector was evaluated. An E.G7 tumor model (Wang, L. andD. Baltimore. 2005. supra.) was used in which OVA serves as the tumorantigen.

The tumor cell lines EL4 (C57BL/6J, H-2″, thymoma) and E.G7 (EL4 cellsstably expressing one copy of chicken OVA cDNA) were used for the tumorchallenge of mice. For the tumor protection experiment, B6 mice (CharlesRiver Breeding Laboratories) received a single injection of 50×10⁶ TU ofthe targeting lentivector (FOVA/SVGmu or FUGW/SVGmu) on the right flank.Two weeks later, 5×10⁶ EL4 or E.G7 cells were injected subcutaneouslyinto the left flank of the mice. Tumor size was measured every other dayusing fine calipers and was shown as the product of the two largestperpendicular diameters a×b (mm²). The mice were killed when the tumorsreached 400 mm².

Vaccination with 50×10⁶ TU FOVA/SVGmu completely protected the mice fromthe E.G7 tumor challenge (FIG. 18, left), while tumors grew rapidly inmice receiving a mock vaccination with a lentivector lacking the OVAtransgene (FIG. 18, left). This protection was OVA-specific because thevaccinated mice grew control EL4 tumors that lack expression of OVA(FIG. 18, right), regardless of the lentivector used for immunization.

Example 14 Generation of Anti-Tumor Immunity Tumor Treatment

The anti-tumor immunity generated after an in vivo administration ofDC-targeted lentivector was evaluated where tumor cells were introducedprior to administration of the lentivector. The steps of tumor injectionand lentivector administration were reversed relative to that in Example13 to test whether an established tumor could be eliminated, in a testof “therapeutic vaccination”. To this end, E.G7 tumor cells expressingthe firefly luciferase gene (E.G7.luc) were used to challenge mice,allowing close monitoring of tumor growth kinetics in live animals usingBLI. To facilitate imaging, an albino strain of B6 mice (The JacksonLaboratory) was used. These mice lack pigmentation and therefore havelow background absorption of the luminescence signal. Injection of thesemice with 100×10⁶ TU of FOVA/SVGmu (Example 10) showed a similarresponse to that observed in canonical B6 mice (FIG. 21). E.G7.luc tumorcells (5×10⁶) were implanted subcutaneously in the albino B6 mice. Themice were immunized by FOVA/SVGmu (50×10⁶ TU per mice per time) twice ondays 3 and 10 post-tumor challenge via subcutaneous injection. Theexperiment was repeated three times with a representative experimentshown in FIGS. 19 and 20.

The mice receiving the DC-targeting lentivector immunization showed adecline of tumor growth starting at day 9, followed by tumor regressionand a reduction of luminescence below the detection level on day 11(FIGS. 19 and 20). Although minimal tumor recurrence was observed fromday 12 to day 16, mice treated with FOVA/SVGmu were free of disease atthe end of day 18 and thereafter; no tumor relapse was observed for aslong as the experiment ran (>60 days). In contrast, tumors grewprogressively in the mice receiving no treatment and the mice had to beremoved from the experiment after day 16 due to the large size of thetumors. It was a interesting to note that tumor regression was observedstarting at 7 days after the lentivector immunization. The timing oftumor regression correlates well with the kinetics of anantigen-specific immune response induced by vaccination.

Example 15 In Vitro Delivery of Antigen and Maturation Factory by aRecombinant Virus

The success of DC vaccination can depend on the maturation state of DCs(Banchereau, J. and A. K. Palucka. 2005. Nat Rev Immunol 5:296-306;Schuler, G., et al. 2003. Curr Opin Immunol 15: 138-147; Figdor, C. G.,et al. 2004. Nat Med 10: 475-480, each of which is incorporated hereinby reference in its entirety). Therefore, genes can be included in thelentiviral vectors that encode the stimulatory molecules to trigger thedesired DC-maturation. Cytokines that can be used include, but are notlimited to, GM-CSF, IL-4, TNFa, IL-6, and the like. In some embodiments,the maturation agent that is used is the CD40 ligand (CD4OL), which istypically expressed on CD4 T cells and serves as a ligand for the CD40receptor on DCs (Matano, T., et al. 1995. J Gen Virol 76: 3165-3169;Nguyen, T. H., et al. 1998. Hum Gene Ther 9: 2469-2479, each of which isincorporated herein by reference in its entirety). To further manipulateDCs to be a potent vaccine for therapy, a drug-inducible CD40 receptor(iCD40) is adapted into the gene delivery system in some embodiments. Asdescribed elsewhere, iCD40 was designed and consists of a cytoplasmicdomain of CD40 fused to ligand-binding domains and a membrane-targetingsequence (Hanks, B. A., et al. 2005. Nat Med 11: 130-137, which isherein incorporated by reference in its entirety). When iCD40 isexpressed, maturation and activation of DCs is regulated with alipid-permeable, dimerizing drug.

To examine the effect of including DC maturation factors, the cDNAs forovalbumin (OVA, as described in Example 10), GM-CSF, IL-4, TNFa, IL-6and CD4OL are obtained. The iCD40 is constructed as described elsewhere(Hanks, B. A., et al. 2005. supra). Using IRES and 2A-like sequences,multicistronic lentiviral vectors capable of efficiently translating upto four proteins are constructed. This system is adapted to constructlentiviral vectors co-expressing the following genes: OVA and amaturation factor molecule (GM-CSF, IL-4, TNFa, IL-6, CD4OL or iCD40)(FIG. 24a , labeled as “FUOIM”). An exemplary vector sequence isprovided by SEQ ID NO: 7. SVGmu-enveloped lentiviruses are prepared (asdescribed in Example 2), and the lentiviruses are transduced • in vitrointo cultured mouse BMDCs (generated as described in Example 6) tospecifically deliver these genes into the cells. Maturation of BMDCs ismeasured by FACS analysis for up-regulation of several key moleculesthat have essential roles in the process of T cell stimulation. Typicalrepresentative markers are ICAM-1 (CD54), B7.1 (CD80), MHC class I, MHCclass II and endogenous CD40. BMDCs transduced with lentivirusesencoding only OVA and GFP genes serve as controls for the experiment. Itis observed that up-regulation of maturation markers is achieved wheniCD40-modified DCs are exposed to an effective amount of dimeric drugAP20187.

In addition, two characteristic features of matured DCs are the reducedcapacity for endocytosis and the improved potential for T cellactivation. The uptake of FITC-tagged dextran is used to quantify theendocytosis of transduced DCs. The mature DCs are also used to stimulateT cells expressing OT1 T cell receptors (TCRs) (as described in Example10), in order to evaluate their capacity to mount an immune response. Itis observed that when iCD40-modified DCs are exposed to an effectiveamount of dimeric drug AP20187, the uptake of FITC-tagged dextran isreduced relative to that of non-iCD40-modified DCs. Furthermore, it isobserved that after coculture with varying ratios of iCD40-modified DCs(treated with the dimeric drug) to transgenic T cells, OT1 T cellsrespond more vigorously as measured by the release of IFN-y and T cellproliferation than do those co-cultured with non-iCD40-modified DCs.

Longevity of DCs is another parameter that determines T-cell-dependentimmunity. The effects of stimulator molecules on DC survival using an invitro serum-starvation assay will be compared using the method asdescribed in Hanks et al. (Hanks, B. A., et al. 2005. supra).

If necessary, two maturation factor molecules can be delivered bylentiviral vector to targeted DCs, as the vector configuration has thecapacity to express four proteins.

Example 16 In Vivo Delivery of Antigen and Maturation Factors by aRecombinant Virus

Recombinant viruses packaged with FUOIM lentiviral vector (SEQ ID NO: 7)are prepared as described in Example 15. The viruses are administered tonaive B6 mice to deliver OVA antigen and maturation factor molecules toDCs, and induction of immunity to graded doses of viruses is evaluatedas described in Example 11.

It is observed that the targeted DC immunization by iCD40-containinglentiviruses increases the number of OVA responsive T cells as comparedto the control mice, which are either not immunized, immunized with anon-OVA containing lentivirus (e.g. FUGW/SVGmu), or immunized withnon-iCD40 containing lentivirus (e.g. FOVA/SVGmu).

In addition, the resistance of the animals to a tumor challenge isassessed with the iCD40-containing lentivectors, as described in Example13. The mice are injected with the following lentivectors in the tumorchallenge experiment: FUOIM/SVGmu, FOVA/SVGmu, or FUGW/SVGmu. Thefollowing cell lines are used for tumor challenge: EL4 (C57BL/6J, H-2¹⁾,thymoma) and E.G7 ((EL4 cells stably expressing one copy of chicken OVAcDNA). It is observed that the mice receiving immunization by theDC-targeting lentivectors FUOIM/SVGmu and FOVA/SVGmu are protected fromthe tumor challenge. In contrast, it is observed that tumors growrapidly in mice receiving a mock vaccination with a lentivector lackingthe OVA transgene (FUGW/SVGmu). This protection is OVA-specific becausethe vaccinated mice grow control EL4 tumors that lack expression of OVA,regardless of the lentivector used for immunization.

Finally, the potential of this method to eradicate an established tumoris assessed with the iCD40-containing lentivectors, as described inExample 14. The following lentivectors are used for immunization in theexperiment: FUOIM/SVGmu and FOVA/SVGmu. The following cell lines areused for tumor treatment: EL4 and E.G7. It is observed that the tumorcell-injected mice receiving immunization by the DC-targetinglentivectors (FUOIM/SVGmu and FOVA/SVGmu) show a decline of tumorgrowth, followed by tumor regression and a reduction of luminescencebelow the detection level. Further, no tumor relapse is observed for aslong as the experiment runs (>60 days). In contrast, tumors growprogressively in the mice receiving no treatment.

Example 17 HIV/AIDS Antigen Presentation by Recombinant Virus In Vitro

To treat HIV/AIDS, “dual-functional” DCs are generated based on thedescribed gene delivery strategy. The “dual functional” DCs areefficacious at both eliciting neutralizing antibodies (Nabs) andinducing T cell immunity (FIG. 25). To efficiently elicit NAbs, a geneencoding chimeric membrane-bound gp120 (gp120 m) is delivered to DCs.Gp120 is an envelope glycoprotein for HIV and is considered to be themost potent immunogen (Klimstra, W. B., et al. 2003. J Virol77:12022-12032; Bernard, K. A., et al. 2000. Virology 276:93-103;Byrnes, A. P., et al. 1998. J Virol 72: 7349-7356, each of which isincorporated herein by reference in its entirety). As described elsehere, gp1 20 fused with the transmembrane domain of the vesicularstomatitis virus glycoprotein can be expressed on the cell surface in atrimeric form, mimicking the mature trimer on the HIV virion surface(Klimstra, W. B., et al. 1998. J Virol 72: 7357-7366, which isincorporated herein by reference in its entirety). This form ofimmunogen will be displayed on the DC's surface. In addition to surfaceexpression, the DCs can also present epitope peptides derived from gpl20 in MHC restricted fashion to T cells.

Since HIV infection can significantly impair DC function through thedepletion of CD4 T cells, it is desirable to engineer DCs that functionindependently of T cells. Expression of CD4OL or iCD40 can result inmaturation and activation of DCs in the absence of CD4 T cells. Thus,the engineered CD4OL or iCD40, as described in Example 16, whichfunctions as a maturation and stimulatory molecule, is incorporated intothe DC-targeting virus.

The lentiviral construct for genetically modifying DCs is illustrated inFIG. 24b and is labeled as FUGmID (SEQ ID NO: 8). Codon-optimized cDNAsfor gp120 from NIH AIDS Research & Reference Reagent Program areobtained. The codon-optimized sequence can achieve exceptionally highlevels of gene expression outside of the context of the HIV-I genome.The construct is prepared by fusion of gp120 with the transmembranedomain of the vesicular stomatitis virus glycoprotein.

In vitro assays are conducted to assess the efficacy of gene-modifiedDCs to elicit NAbs. CD19⁺ B cells are isolated from the spleens of naiveB6 mice using anti-CD19 microbeads (MiHenyi Biotech, Auburn, Calif.) andco-cultured with modified DCs in the presence of IL-4 and IL-6. Thelentiviral vector FUmGID is co-transfected with SVGmu in cell lines toprepare the FUmGID/SVGmu virus, as described in Example 2. The resultantviruses are transduced into bone marrow-derived DCs (BMDCs). Thetransduced DCs are be irradiated (3,000 rad) and used as antigenpresenting cells (APCs) in co-culture with B cells. The time course ofthe proliferation of B cells in response to transduced BMDCs ismeasured. It is observed that B cells proliferate to a greater extent inco-culture with transduced BMDCs than those that are co-cultured withmock-transduced BMDCs.

To investigate the effect of genetically modified DCs on thedifferentiation of B cells into specific immunoglobulin-secreting cells,the co-culture method as previously described is employed with theexception that the DCs are not irradiated. After 14 days, the titer ofvarious isotypes of HIV-specific antibody in culture supernatants isdetermined by ELISA using recombinant gpI20 (available from NIB: AIDSResearch & Reference Reagent Program) as the antigen. Expression of thevarious isotypes of HIV-specific antibody are greater in B-cellsco-cultured with transduced BMDCs than in those cocultured withmock-transduced BMDCs.

To assess the efficacy of the genetically modified DCs to activate Tcells in vitro, CD3⁺ T cells are isolated from naive B6 mice andco-cultured with lentivirus-infected and irradiated DCs. The time courseof T cell proliferation is measured. T cell proliferation is found to begreater in T cell cultures co-cultured with transduced and irradiatedDCs than in those co-cultured with mock-transduced DCs.

The results are expected to collectively demonstrate that BMDCstransduced with the FUmGID/SVGmu lentivector is effective in bothstimulation of B-cells to produce neutralizing antibodies (Nabs) and ininducing T cell immunity against HIV/AIDS.

Example 18 HIV/AIDS Antigen Presentation by Recombinant Virus In Vivo

To evaluate the activation of B cells in vivo, B6 mice are immunized bysubcutaneous injection with the recombinant lentiviruses prepared asdescribed in Example 17. Controls include mice injected withlentiviruses encoding antigens alone, lentiviruses encoding maturationmolecules alone, and naive mice without any treatment. Two weeks aftervirus injection, serum antibodies against HIV are measured by ELISA. Theantibody titer is found to be higher in those mice injected with theFUmGID/SVGmu virus as well as in those injected with lentivirus encodingantigens alone. In contrast. antibody titer is relatively low in thosemice immunized with lentivirus encoding maturation molecules alone andin naïve mice.

For in vivo activation of T cells, the recombinant viruses described areinjected into B6 mice. Seven days later, T cells are isolated, and theirproliferation and cytokine secretion, after in vitro restimulation withgenetically modified DCs, is measured as described in Example 12. Thedurability of the effector T cell responses is also monitored.Lentivector targeting to native DCs is able to elicit HIV-responsive Tcells in both the lymph node and spleen. Administration of recombinantFUmGID/SVGmu is sufficient to generate T cells which secrete IFN-y. Incontrast, administration of a mock control vector (e.g. FUGW/SVGmu)fails to elicit an HIV-specific response.

Example 19 In Situ HIV/AIDS Vaccination by Recombinant Virus ProtectionAgainst HIV Challenge

In order to test in situ DC vaccination approach to deal with HIV, a newmouse model of HIV pathogenesis involving human/mouse chimeras isdeveloped. As described elsewhere, the RAG2^(−/−)y,^(−/−) mouse can bereconstituted with a human adaptive immune system (Strauss, J. H., etal. 1994. Archives of Virology 9:473-484, which is incorporated hereinby reference in its entirety). The RAG2⁴⁻y_(c) ^(−/−) mice lack B, T,and NK cells (Morizono, K., et al. 2001. J Virol 75: 8016-8020, which isincorporated herein by reference in its entirety). Injection of CD34⁺human cord blood into the liver of one-day old partially-irradiated miceleads to the generation and maturation of functionally diverse humanDCs, B cells, and T cells with human MHC restriction. Additionally, thismodel directs the development of primary and secondary lymphoid organs,and the production of a functional CD8⁺ T cell immune response against aviral challenge. Furthermore, the observation of the Ig isotypeswitching from IgM to IgG indicates the existence of functional CD4⁺ Tcell immunity.

To determine the effectiveness of preventive protection against HIV byDC-targeted immunization, the human/mouse chimeras are administeredrecombinant viruses enveloped with SVGmu by injection. The recombinantviruses encode gp120 m antigen (Example 17) in conjunction with amaturation stimulator (for example, CD4OL or iCD40 as in Example 15),and they are prepared and concentrated as described in Example 2. Theimmunized mice are then inoculated with HIV according to methods wellknown in the art, such as, for example, via intraperitoneal orintravenous routes. Since the reconstituted mice maintain human CD4 Tcells, the animals are challenged with molecularly cloned HIV reporterviruses, NFNSX-r-HSAS (CCR5-tropic), NL-r-HSAS (CXCR4-tropic) andclinical isolates (Baenziger, et al. 2006. Proc Natl Acad Sci USA103:15951-15956, which is incorporated herein by reference in itsentirety). The replication-competent reporter viruses also contain theheat-stable antigen (HSA) in the vpr region. Further, to establish aproductive infection prior to inoculation, infected syngeneic peripheralblood mononuclear cells (PBMCs) are injected into the peritoneal spaceof the reconstituted human/mouse chimera.

Evidence of HIV infection is monitored over time in spleens, lymphnodes, PBMCs, and peripheral blood. FACS for HSA in the HIV reporterviruses is used to test for HIV viral integration and replication. HIVviral load is also measured from plasma using RT-PCR. Through evaluationof HIV infection by these methods, it is observed that productive insitu DC vaccination makes the immunized mice more resistant to the HIVchallenge than those which are not immunized.

Example 20 In Situ HIV/AIDS Vaccination by Recombinant Virus Clearanceof HIV Infection

To test the ability of the in situ DC vaccination approach to clear anactive HIV infection, human/mouse chimeras are first challenged withmolecularly cloned HIV reporter virus, NFNSX-r-HSAS (CCR5-tropic), asdescribed in Example 19. Active HIV infection is monitored by FACSanalysis of HSA expression in human CD4 T cells. Once successful HIVinfection is confirmed, the engineered recombinant viruses (Example 19)are injected into animals via subcutaneous injection or by an optimalroute determined by one of skill in the art (for example, s.c., i.d.,i.v. or i.p.). The HIV viral load is then monitored by RT-PCR, andperipheral CD4 counts are followed. It is observed that DC vaccinationis able to lower HIV viral load and to clear an established HIVinfection in immunized mice compared to non-vaccinated controls.

Highly active antiretroviral therapy (HAART), utilizing a three-drugstrategy, has significantly improved AIDS morbidity and mortality. Thestrategy outlined above can be adapted to this paradigm bysimultaneously transducing DC cells in vivo with engineered recombinantviruses. In conjunction with HAART, the above studies are repeated toevaluate the ability to prevent or reduce infection after HIV challenge(Example 19) and to clear an active HIV infection.

Example 21 Treatment of a Malignant Tumor in a Human Using a RecombinantVirus

A human patient is diagnosed with a malignant tumor. The patient isadministered a suitable amount of recombinant virus containing a genethat encodes an antigen specific for the tumor and enveloped with aDC-SIGN specific targeting molecule, such as, for example, SVGmu. Thevirus optionally contains a gene encoding a DC maturation factor, asdescribed in Example 15. The virus is administered by weekly intravenousinjection for the duration of treatment. At periodic times during andafter the treatment regimen, tumor burden is assessed by magneticresonance imaging (MRI). Significant reductions in tumor size are foundas treatment progresses.

Example 22 Prevention of Tumor Formation in a Human Using a RecombinantVirus

A group of human patients is administered a suitable amount ofrecombinant virus containing at least one gene encoding an antigen thatis commonly and specifically associated with tumor cells and optionallycontaining a gene encoding a DC maturation factor, as described inExample 15. The virus is enveloped with a DC-SIGN specific targetingmolecule, such as, for example, SVGmu (Example 2). Patients in theexperimental group and in a control group are monitored periodically fortumor growth. It is observed that the incidence of malignant tumorformation is lower in patients to whom the virus is administered than inthe control group.

Example 23 Treatment of AIDS/HIV in a Human Using a Recombinant Virus

A human patient is diagnosed with HIV/AIDS. The patient is administereda suitable amount of recombinant virus containing a gene that encodesGp120 (Example 17) and enveloped with a DC-SIGN specific targetingmolecule, such as, for example, SVGmu (Example 2). The virus optionallycontains a gene encoding a DC maturation factor, as described in Example15. The virus is administered by weekly intravenous injection for theduration of treatment. At periodic times during and after the treatmentregimen, HIV viral load is assessed by measuring antibodies in thepatient's blood against HIV using ELISA. The patient's T-cell count isalso evaluated. It is observed that a significant reduction in HIV viralload is achieved as treatment progresses. Furthermore, it is observedthat the patient's T-cell count stops decreasing as treatmentprogresses.

Example 24 Prevention of HIV/AIDS in a Human Using a Recombinant Virus

A group of human patients considered at risk for HIV infection isadministered a suitable amount of recombinant virus containing a geneencoding GP120 (Example 17) and optionally containing a gene encoding aDC maturation factor, as described in Example 15. The virus is envelopedwith a DC-SIGN specific targeting molecule, such as, for example, SVGmu(Example 2). Patients in the experimental group and in a control groupare tested every 6 months for HIV infection and if positive, monitoredfor HIV viral load and T-cell counts. In positively infected patientswithin the vaccinated group, it is observed that HIV viral load stayslow and T-cell counts remain high relative to the positively-infectedpatients of the control group.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

What is claimed is:
 1. A method of stimulating an antigen-specificimmune response in a mammal comprising: administering to said mammal arecombinant replication deficient lentivirus pseudotyped with a modifiedE2 alphavirus glycoprotein that transduces dendritic cells moreefficiently compared to other cell types, wherein the lentiviruscomprises an exogenous polynucleotide encoding an antigen associatedwith a disease, and wherein said lentivirus is administered in an amountsufficient to stimulate an immune response to said antigen.
 2. Themethod of claim 1 wherein the immune response is an antibody response orantigen-specific T-cell response or both.
 3. The method of claim 2wherein the immune response is an antigen-specific T-cell response. 4.The method of claim 3 wherein the antigen-specific T-cell response is aCD8⁺ T-cell response.
 5. The method of claim 1 wherein the virusenvelope further comprises an E1 alphavirus glycoprotein.
 6. The methodof claim 1 wherein the E2 glycoprotein is produced from the nucleic acidsequence of SEQ ID NO: 3 or comprises the amino acid sequence of SEQ IDNO:
 11. 7. The method of claim 1 wherein the lentivirus comprises an HIVvector, a MSCV vector or an MLV vector.
 8. The method of claim 1 whereinthe replication deficient lentivirus further comprises one or moreadditional polynucleotides of interest.
 9. The method of claim 1 whereinthe replication deficient lentivirus further comprises a polynucleotideencoding a second antigen associated with a disease.
 10. The method ofclaim 1 wherein the mammal is a human.
 11. The method of claim 1 whereinthe antigen is a tumor associated antigen, viral antigen, bacterialantigen, fungal antigen, protozoan parasite antigen, helminth parasiteantigen, or ectoparasite antigen.
 12. The method according to claim 11wherein the antigen is Her-2 receptor, MAGE, BAGE, RAGE, NY-ESO,MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase, tyrosinase-relatedprotein, prostate specific membrane antigen (PSMA), prostate-specificantigen (PSA), ras, bcr/ab1, Her2/neu, p53, cytochrome P450 1B1,N-acetylglucosaminyltransferase-V, human papilloma virus protein E6,human papilloma virus protein E7, carcinoembryonic antigen andalpha-fetoprotein, gp120, an adenovirus polypeptide, an alphaviruspolypeptide, a calicivirus polypeptide, calicivirus capsid antigen, acoronavirus polypeptide, s distemper virus polypeptide, an Ebola viruspolypeptide, an enterovirus polypeptide, a flavivirus polypeptides, ahepatitis virus (AE) polypeptide, a hepatitis B core antigen, ahepatitis or surface antigen, a herpesvirus polypeptide, a herpessimplex virus glycoptrotein, a varicella zoster virus glycoprotein, animmunodeficiency virus polypeptide, a human immunodeficiency virusenvelope protein, a human immunodeficiency virus protease, an infectiousperitonitis virus polypeptide, an influenza virus polypeptide, aninfluenza A hemagglutinin, an influenza A neuraminidase, an influenza Anucleoprotein, a leukemia virus polypeptide, a Marburg viruspolypeptide, an orthomyxovirus polypeptide, a papilloma viruspolypeptide, a parainfluenza virus polypeptide, a parainfluenza virushemagglutinin, aparainfluenza virus neuraminidase, a paramyxoviruspolypeptide, a parvovirus polypeptide, a pestivirus polypeptide, apicorna virus polypeptidea poliovirus capsid polypeptide, a pox viruspolypeptide, a vaccinia virus polypeptide, a rabies virus polypeptide, arabies virus glycoprotein G, a reovirus polypeptide, a retroviruspolypeptide, a rotavirus polypeptide, an Absidia polypeptide, anAcremonium polypeptide, an Alternaria polypeptide, an Aspergilluspolypeptide, a Basidiobolus polypeptide, a Bipolaris polypeptide, aBlastomyces polypeptide, a Candida polypeptide, a Coccidioidespolypeptide, a Conidiobolus polypeptide, a Cryptococcus polypeptide, aCurvalaria polypeptide, an Epidermophyton polypeptide, an Exophialapolypeptide, an Geotrichum polypeptide, a Histoplasma polypeptide, aMadurella polypeptide, a Malassezia polypeptide, a Microsporumpolypeptide, a Moniliella polypeptide, a Mortierella polypeptide, aMucor polypeptide, a Paecilomyces polypeptide, a Penicilliumpolypeptide, a Phialemonium polypeptide, a Phialophora polypeptide, aPrototheca polypeptide, a Pseudallescheria polypeptide, aPseudomicrodochium polypeptide, a Pythium polypeptide, a Rhinosporidiumpolypeptide, a Rhizopus polypeptide, a Scolecobasidium polypeptide, aSporothrix polypeptide, a Stemphylium polypeptide, a Trichophytonpolypeptide, a Trichosporon polypeptide, and a Xylohypha polypeptide, aBabesia polypeptide, a Balantidium polypeptide, a Besnoitia polypeptide,a Cryptosporidium polypeptide, an Eimeria polypeptide, anEncephalitozoon polypeptide, an Entamoeba polypeptide, a Giardiapolypeptide, a Hammondia polypeptide, a Hepatozoon polypeptide, anIsospora polypeptide, a Leishmania polypeptide, a Microsporidiapolypeptide, a Neospora polypeptide, a Nosema polypeptide, aPentatrichomonas polypeptide, a Plasmodium polypeptide, P. falciparumcircumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2),carboxyl terminus of liver state antigen 1 (PfLSA1 c-term), exportedprotein 1 (PfExp-1), a Pneumocystis polypeptide, a Sarcocystispolypeptide, a Schistosoma polypeptide, Ta heileria polypeptide, aToxoplasma polypeptide, a Trypanosoma polypeptide, an Acanthocheilonemapolypeptide, an Aelurostrongylus polypeptide, an Ancylostomapolypeptide, an Angiostrongylus polypeptide, an Ascaris polypeptide, aBrugia polypeptide, a Bunostomum polypeptide, a Capillaria polypeptide,a Chabertia polypeptide, a Cooperia polypeptide, a Crenosomapolypeptide, a Dictyocaulus polypeptide, a Dioctophyme polypeptide, aDipetalonema polypeptide, a Diphyllobothrium polypeptide, a Diplydiumpolypeptide, a Dirofilaria polypeptide, a Dracunculus polypeptide, anEnterobius polypeptide, a Filaroides polypeptide, a Haemonchuspolypeptide, a Lagochilascaris polypeptide, a Loa polypeptide, aMansonella polypeptide, a Muellerius polypeptide, a Nanophyetuspolypeptide, a Necator polypeptide, a Nematodirus polypeptide, anOesophagostomum polypeptide, an Onchocerca polypeptide, an Opisthorchispolypeptide, an Ostertagia polypeptide, a Parafilaria polypeptide, aParagonimus polypeptide, a Parascaris polypeptide, a Physalopterapolypeptide, a Protostrongylus polypeptide, a Setaria polypeptide, aSpirocerca polypeptide, a Spirometra polypeptide, a Stephanofilariapolypeptide, a Strongyloides polypeptide, a Strongylus polypeptide, aThelazia polypeptide, a Toxascaris polypeptide, a Toxocara polypeptide,a Trichinella polypeptide, a Trichostrongylus polypeptide, a Trichurispolypeptide, an Uncinaria polypeptide, a Wuchereria polypeptide, a fleapolypeptide, a tick polypeptide, a hard tick polypeptide, a soft tickpolypeptide, a fly polypeptide, a midge polypeptide, a mosquitopolypeptide, a sand fly polypeptide, a black fly polypeptide, a horsefly polypeptide, a horn fly polypeptide, a deer fly polypeptide, atsetse fly polypeptide, a stable fly polypeptide, a myiasis-causing flypolypeptide, a biting gnat polypeptide, an ant polypeptide, a spiderpolypeptide, a lice polypeptide, a mite polypeptide, a true bugpolypeptide, a bed bug polypeptide, and a kissing bug polypeptide. 13.The method of claim 1 wherein the lentivirus further comprises anucleotide sequence encoding a maturation factor.
 14. The method ofclaim 13 wherein the maturation factor is selected from the groupconsisting of GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFα,B7.1, B7.2, 4-1BB, CD40 ligand (CD40L) and drug-inducible CD40 (iCD40).15. The method of claim 1 wherein the replication deficient lentivirusis administered subcutaneously or intradermally.
 16. The method of claim1 wherein the E2 alphavirus glycoprotein is a Sindbis virusglycoprotein.
 17. The method of claim 16 wherein the replicationdeficient lentivirus is administered subcutaneously or intradermally.18. The method of claim 16 wherein the lentivirus comprises an HIVvector, a MSCV vector or an MLV vector.
 19. The method of claim 16wherein the replication deficient lentivirus further comprises apolynucleotide encoding a second antigen associated with a disease. 20.The method of claim 16 wherein the mammal is a human.
 21. The method ofclaim 16 wherein the antigen is a tumor associated antigen, viralantigen, bacterial antigen, fungal antigen, protozoan parasite antigen,helminth parasite antigen, or ectoparasite antigen.
 22. The methodaccording to claim 21 wherein the antigen is Her-2 receptor, MAGE, BAGE,RAGE, NY-ESO, MART-1/Melan-A, gp100, gp75, mda-7, tyrosinase,tyrosinase-related protein, prostate specific membrane antigen (PSMA),prostate-specific antigen (PSA), ras, bcr/ab1, Her2/neu, p53, cytochromeP450 1B1, N-acetylglucosaminyltransferase-V, human papilloma virusprotein E6, human papilloma virus protein E7, carcinoembryonic antigenand alpha-fetoprotein, gp120, an adenovirus polypeptide, an alphaviruspolypeptide, a calicivirus polypeptide, calicivirus capsid antigen, acoronavirus polypeptide, s distemper virus polypeptide, an Ebola viruspolypeptide, an enterovirus polypeptide, a flavivirus polypeptides, ahepatitis virus (AE) polypeptide, a hepatitis B core antigen, ahepatitis or surface antigen, a herpesvirus polypeptide, a herpessimplex virus glycoptrotein, a varicella zoster virus glycoprotein, animmunodeficiency virus polypeptide, a human immunodeficiency virusenvelope protein, a human immunodeficiency virus protease, an infectiousperitonitis virus polypeptide, an influenza virus polypeptide, aninfluenza A hemagglutinin, an influenza A neuraminidase, an influenza Anucleoprotein, a leukemia virus polypeptide, a Marburg viruspolypeptide, an orthomyxovirus polypeptide, a papilloma viruspolypeptide, a parainfluenza virus polypeptide, a parainfluenza virushemagglutinin, aparainfluenza virus neuraminidase, a paramyxoviruspolypeptide, a parvovirus polypeptide, a pestivirus polypeptide, apicorna virus polypeptidea poliovirus capsid polypeptide, a pox viruspolypeptide, a vaccinia virus polypeptide, a rabies virus polypeptide, arabies virus glycoprotein G, a reovirus polypeptide, a retroviruspolypeptide, a rotavirus polypeptide, an Absidia polypeptide, anAcremonium polypeptide, an Alternaria polypeptide, an Aspergilluspolypeptide, a Basidiobolus polypeptide, a Bipolaris polypeptide, aBlastomyces polypeptide, a Candida polypeptide, a Coccidioidespolypeptide, a Conidiobolus polypeptide, a Cryptococcus polypeptide, aCurvalaria polypeptide, an Epidermophyton polypeptide, an Exophialapolypeptide, an Geotrichum polypeptide, a Histoplasma polypeptide, aMadurella polypeptide, a Malassezia polypeptide, a Microsporumpolypeptide, a Moniliella polypeptide, a Mortierella polypeptide, aMucor polypeptide, a Paecilomyces polypeptide, a Penicilliumpolypeptide, a Phialemonium polypeptide, a Phialophora polypeptide, aPrototheca polypeptide, a Pseudallescheria polypeptide, aPseudomicrodochium polypeptide, a Pythium polypeptide, a Rhinosporidiumpolypeptide, a Rhizopus polypeptide, a Scolecobasidium polypeptide, aSporothrix polypeptide, a Stemphylium polypeptide, a Trichophytonpolypeptide, a Trichosporon polypeptide, and a Xylohypha polypeptide, aBabesia polypeptide, a Balantidium polypeptide, a Besnoitia polypeptide,a Cryptosporidium polypeptide, an Eimeria polypeptide, anEncephalitozoon polypeptide, an Entamoeba polypeptide, a Giardiapolypeptide, a Hammondia polypeptide, a Hepatozoon polypeptide, anIsospora polypeptide, a Leishmania polypeptide, a Microsporidiapolypeptide, a Neospora polypeptide, a Nosema polypeptide, aPentatrichomonas polypeptide, a Plasmodium polypeptide, P. falciparumcircumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2),carboxyl terminus of liver state antigen 1 (PfLSA1 c-term), exportedprotein 1 (PfExp-1), a Pneumocystis polypeptide, a Sarcocystispolypeptide, a Schistosoma polypeptide, Ta heileria polypeptide, aToxoplasma polypeptide, a Trypanosoma polypeptide, an Acanthocheilonemapolypeptide, an Aelurostrongylus polypeptide, an Ancylostomapolypeptide, an Angiostrongylus polypeptide, an Ascaris polypeptide, aBrugia polypeptide, a Bunostomum polypeptide, a Capillaria polypeptide,a Chabertia polypeptide, a Cooperia polypeptide, a Crenosomapolypeptide, a Dictyocaulus polypeptide, a Dioctophyme polypeptide, aDipetalonema polypeptide, a Diphyllobothrium polypeptide, a Diplydiumpolypeptide, a Dirofilaria polypeptide, a Dracunculus polypeptide, anEnterobius polypeptide, a Filaroides polypeptide, a Haemonchuspolypeptide, a Lagochilascaris polypeptide, a Loa polypeptide, aMansonella polypeptide, a Muellerius polypeptide, a Nanophyetuspolypeptide, a Necator polypeptide, a Nematodirus polypeptide, anOesophagostomum polypeptide, an Onchocerca polypeptide, an Opisthorchispolypeptide, ab Ostertagia polypeptide, a Parafilaria polypeptide, aParagonimus polypeptide, a Parascaris polypeptide, a Physalopterapolypeptide, a Protostrongylus polypeptide, a Setaria polypeptide, aSpirocerca polypeptide, a Spirometra polypeptide, a Stephanofilariapolypeptide, a Strongyloides polypeptide, a Strongylus polypeptide, aThelazia polypeptide, a Toxascaris polypeptide, a Toxocara polypeptide,a Trichinella polypeptide, a Trichostrongylus polypeptide, a Trichurispolypeptide, an Uncinaria polypeptide, a Wuchereria polypeptide, a fleapolypeptide, a tick polypeptide, a hard tick polypeptide, a soft tickpolypeptide, a fly polypeptide, a midge polypeptide, a mosquitopolypeptide, a sand fly polypeptide, a black fly polypeptide, a horsefly polypeptide, a horn fly polypeptide, a deer fly polypeptide, atsetse fly polypeptide, a stable fly polypeptide, a myiasis-causing flypolypeptide, a biting gnat polypeptide, an ant polypeptide, a spiderpolypeptide, a lice polypeptide, a mite polypeptide, a true bugpolypeptide, a bed bug polypeptide, and a kissing bug polypeptide. 23.The method of claim 16 wherein the lentivirus further comprises anucleotide sequence encoding a maturation factor.